Chloride salt of tat-nr2b9c

ABSTRACT

The present invention provides lyophilized formulations of active agents, particularly of TAT-NR2B9c, as chloride salts. TAT-NR2B9c has shown promise for treating stroke, aneurysm, subarachnoid hemorrhage and other neurological or neurotraumatic conditions. The chloride salt of TAT-NR2B9c shows improved stability compared with the acetate salt form of prior formulations. Formulations of the chloride salt of TAT-NR2B9c are stable at ambient temperature thus facilitating maintenance of supplies of such a formulation in ambulances for administration at the scene of illness or accident or in transit to a hospital.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/313,940 filed Nov. 23, 2015, which is a US National Stage ofPCT/IB2015/053995 filed May 28, 2015, which claims priority to U.S.Provisional Application No. 62/004,142 filed May 28, 2014, each of whichis hereby incorporated by reference in its entirety for all purposes.

REFERENCE TO SEQUENCE LISTING

This application includes an electronic sequence listing in a file named520184_SEQLST.TXT, created on Sep. 11, 2018 and containing 16,738 bytes,which is hereby incorporated by reference.

BACKGROUND

Tat-NR2B9c (also known as NA-1) is an agent that inhibits PSD-95, thusdisrupting binding to N-methyl-D-aspartate receptors (NMDARs) andneuronal nitric oxide synthases (nNOS) and reducing excitoxicity inducedby cerebral ischemia. Treatment reduces infarction size and functionaldeficits. TAT-NR2B9c has undergone a successful phase II trial (see WO2010144721 and Aarts et al., Science 298, 846-850 (2002), Hill et al.,Lancet Neurol. 11:942-950 (2012)).

Because TAT-NR2B9c is free of serious side effects, it can beadministered when stroke or other ischemic conditions or hemorrhagicconditions is suspected without a diagnosis according to art-recognizedcriteria having been made to confirm that no hemorrhage is present. Forexample, TAT-NR2B9c can be administered at the location where the strokeor neurotrauma has occurred (e.g., in the patients' home) or in anambulance transporting a subject to a hospital.

TAT-NR2B9c has previously been described as a liquid composition ofnormal saline or phosphate buffered saline or a lyophilized compositionfrom normal saline (WO2010144721).

SUMMARY OF THE CLAIMED INVENTION

The invention provides a chloride salt of a peptide which is TAT-NR2B9c(SEQ ID NO:6) or differs from TAT-NR2B9c by up to 5 amino acidsubstitutions, insertions or deletions, or any other peptide disclosedas an active agent herein. The chloride salt can be prepared byexchanging trifluoroacetate for chloride in a trifluoroacetate salt ofTAT-NR2B9c. The chloride salt can also be prepared by exchangingtrifluoroacetate for acetate and then acetate for chloride staring froma trifluoroacetate salt of TAT-NR2B9c. Optionally, greater than 99% ofanions in the salt are chloride.

The invention further provides a prelyophilized formulation comprising achloride salt as described above a buffer and a sugar. Optionally, thechloride salt is a chloride salt of TAT-NR2B9c. Optionally, the bufferis histidine and the sugar is trehalose and the pH is 6-7. Optionally,acetate and trifluoroacetate each comprise less than 1% of anions byweight in the formulation. Optionally, acetate and trifluoroacetate eachcomprise less than 0.1% by weight of anions in the formulation.Optionally, the chloride salt of the peptide is at a concentration of70-120 mg/ml, the histidine is at a concentration of 15-100 mM, and thetrehalose is at a concentration of 80-160 mM. Optionally, the chloridesalt of the peptide is at a concentration of 70-120 mg/ml, the histidineis at a concentration of 20-100 mM, and the trehalose is at aconcentration of 100-140 mM. Optionally, the Tat-NR2B9c is at aconcentration of 70-120 mg/ml, the concentration of histidine 20-50 mM,and the concentration of trehalose is 100-140 mM. Optionally, theconcentration of histidine is 20 mM and the concentration of trehaloseis 100-200 mM, preferably 120 mM and the concentration of TAT-NR2B9c is90 mg/ml.

The invention further provides a lyophilized formulation prepared bylyophilizing any of the prelyophilized formulations described above.Optionally, acetate and trifluoroacetate each comprise less than 1% byweight of anions in the formulation. Optionally, acetate andtrifluoroacetate each comprise less than 0.1% by weight of anions in theformulation.

The invention further provides a reconstituted formulation prepared bycombining any of the lyophilized formulations described above with anaqueous solution. Optionally, the aqueous solution is water or normalsaline. Optionally, the volume of the reconstituted formulation is 3-6times the volume of the prelyophilized formulation.

The invention further provides a reconstituted formulation comprisingTAT-NR2B9c or other active agent described herein at concentration of15-25 mg/ml, a buffer and a sugar. Optionally, the buffer is histidineat a concentration of 4-20 mM and the sugar is trehalose at aconcentration of 20-30 mM and the pH is 6-7. Optionally, thereconstituted formulation of claim 19 wherein acetate andtrifluoroacetate each comprise less than 1% by weight of anions in theformulation. Optionally, acetate and trifluoroacetate each comprise lessthan 0.1% by weight of anions in the formulation.

The invention further provides a method of preparing a formulation,comprising storing a lyophilized formulation sample as described abovefor at least a week at a temperature of at least 20° C.; andreconstituting the lyophilized formulation. Optionally the lyophilizedformulation is reconstituted in water or saline. Optionally, the methodalso includes administering the reconstituted formulation, optionallyafter further dilution in normal saline, to a patient. Optionally, theformulation is stored for at least a year. Optionally, the storage is atambient temperature. Optionally, the storage includes periods in whichthe temperature exceeds 37° C. In some methods, the patient has strokeor traumatic injury to the CNS. In some methods, the lyophilized sampleis stored in an ambulance. In some methods, the patient has asubarachnoid hemorrhage. In some methods, the patient is undergoingendovascular repair for an aneurysm.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1: Graph shows the infarct area of the rat brain after 3PVO strokefollowing treatment with different formulations of TAT-NR2B9c.

FIGS. 2A, B: A) Bar graph demonstrating the stability of differentTAT-NR2B9c formulations at −20° C. and 40° C. Y axis represents purityof the TAT-NR2B9c after 1 week at the storage temperature as measured by% total area using RP-HPLC. B) same data as A, but sorted by buffer andpH.

FIG. 3: Bar graph demonstrating the stability (by HPLC) of 20 mg/mlTAT-NR2B9c in Histidine buffer, pH 6.5, in the presence of differentbulking agents and salt at −20° C. and 40° C.

FIGS. 4A, B: Differential scanning calorimetry graphs of 20 mg/mlTAT-NR2B9c in histidine buffer pH 6.5 in the presence of Mannitol (A) orMannitol and NaCl (B).

FIGS. 5A, B: Differential scanning calorimetry graphs of 20 mg/mlTAT-NR2B9c in histidine buffer pH 6.5 in the presence of Trehalose (A)or Trehalose and NaCl (B).

FIGS. 6A, B: Differential scanning calorimetry graph of 20 mg/mlTAT-NR2B9c in histidine buffer pH 6.5 in the presence of Dextran-40 (A)or Dextran-40 and NaCl (B).

FIGS. 7A, B: A) Cake appearance following lyophilization of 3 mL of 90mg/ml TAT-NR2B9c in 100 mM Histidine pH 6.5 with 120 mM Trehalose. B).Cake appearance of alternative TAT-NR2B9c formulations with differentamounts of histidine and trehalose.

DEFINITIONS

As well as active ingredients, lyophilized formulations can include oneor more of the following classes of components. The classes are notmutually exclusive; in other words the same agent can component can fallwithin multiple classes.

A “bulking agent” provides structure to a freeze-dried peptide. Bulkingagents include, mannitol, trehalose, dextran-40, glycine, lactose,sorbitol, and sucrose among others. In addition to providing apharmaceutically elegant cake, bulking agents may also impart usefulqualities in regard to modifying the collapse temperature, providingfreeze-thaw protection, glass transition temperature and enhancing theprotein stability over long-term storage. These agents can also serve astonicity modifiers.

A buffer is an agent that maintains the solution pH in an acceptablerange prior to lyophilization. A preferred buffer is histidine. Otherbuffers include succinate (sodium or potassium), histidine, citrate(sodium), gluconate, acetate, phosphate, Tris and the like. Preferredbuffers are effective in a pH range from about 5.5 to about 7 or about 6to about 7; preferably a pH of about 6.5. Examples of buffers thatcontrol the pH in this range include succinate (such as sodiumsuccinate), gluconate, histidine, citrate and other organic acidbuffers.

A “cryoprotectant” provides stability to a peptide againstfreezing-induced stresses, presumably by being preferentially excludedfrom the protein surface. It may also offer protection during primaryand secondary drying, and long-term product storage. Examples arepolymers such as dextran and polyethylene glycol; sugars (includingsugar alcohols) such as sucrose, glucose, trehalose, and lactose; andsurfactants such as polysorbates; and amino acids such as glycine,arginine, and serine.

A lyoprotectant provides stability to the peptide during the drying or‘dehydration’ process (primary and secondary drying cycles), presumablyby providing an amorphous glassy matrix and by binding with the proteinthrough hydrogen bonding, replacing the water molecules that are removedduring the drying process. This helps to maintain the peptideconformation, minimize peptide degradation during the lyophilizationcycle and improve the long-term product stability. Examples includepolyols or sugars such as sucrose and trehalose.

To the extent not already mentioned, other stabilizers or inhibitors ofdegradations can be included deamidation inhibitors, surfactants, somecommon ones are fatty acid esters of sorbitan polyethoxylates (e.g.,polysorbate 20 or polysorbate 80), poloxamer 188, and detergents.

The terms “lyophilization,” “lyophilized,” and “freeze-dried” refer to aprocess by which the material to be dried is first frozen and then theice or frozen solvent is removed by sublimation in a vacuum environment.

A “pharmaceutical formulation” or composition is a preparation thatpermits an active agent to be effective, and lacks additional componentswhich are toxic to the subjects to which the formulation would beadministered.

“Reconstitution time” is the time that is required to rehydrate alyophilized formulation with a solution to solution which is free ofparticles to the naked eye.

A “stable” lyophilized peptide formulation is one with no significantchanges observed at 20° C. for at least one week, month, or morepreferably at least three months, at least six months or a year. Changesare considered insignificant if no more than 10%, preferably 5%, ofpeptide is degraded as measured by SEC-HPLC. The rehydrated solution iscolorless, or clear to slightly opalescent by visual analysis. Theconcentration, pH and osmolality of the formulation have no more than+/−10% change after storage. Potency is within 70-130%, preferably80-120% or sometimes 80-100% of a freshly prepared control sample. Nomore than 10%, preferably 5% of clipping is observed. No more than 10%,preferably 5% of aggregation is formed. Stability can be measured byvarious methods reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, A. Adv. Drug Delivery Rev. 10:29-90 (1993).

The term “isotonic” means that the formulation of interest hasessentially the same osmotic pressure as human blood. Isotonicformulations will generally have an osmotic pressure from about 270-328mOsm. Slightly hypotonic pressure is 250-269 and slightly hypertonicpressure is 328-350 mOsm. Osmotic pressure can be measured, for example,using a vapor pressure or ice-freezing type osmometer.

Tonicity Modifiers: Salts (NaCl, KCl, MgCl2, CaCl2) can be used astonicity modifiers to control osmotic pressure. In addition,cryoprotectants/lyoprotectants and/or bulking agents such as sucrose,mannitol, or glycine can serve as tonicity modifiers.

Numeric values such as concentrations or pH's are given within atolerance reflecting the accuracy with which the value can be measured.Unless the context requires otherwise, fractional values are rounded tothe nearest integer. Unless the context requires otherwise, recitationof a range of values means that any integer or subrange within the rangecan be used.

The terms “disease” and “condition” are used synonymously to indicateany disruption or interruption of normal structure or function in asubject.

DETAILED DESCRIPTION I. General

Peptides synthesized by solid state methods are typically produced astrifluoroacetate salts because trifluoroacetic acid (TFA) is used fordeprotection of peptides and/or removal of peptides from resins. Forpharmaceutical use, the trifluoroacetate is usually replaced withacetate as a counterion because acetate is nontoxic and the replacementof trifluoroacetate by acetate is straightforward. Such has been thecase for the peptide TAT-NR2B9c synthesized to-date as described inWO2010144721 or PCT/US2013/071755 and elsewhere.

Acetate is often a preferred counterion for pharmaceutically peptidesbecause trifluoroacetate can be exchanged for acetate with a minorchange in the typical purification process of a peptide resulting fromsolid phase synthesis in which the final wash is performed with aceticacid instead of trifluoroacetic acid followed by elution withacetonitrile. Occasionally other counterions are used instead. Forexample, chloride is sometimes used for poorly soluble peptides becauseit improves their solubility. However, conversion of trifluoride acetateor acetate to chloride results in loss of some peptide. Moreover,presence of HCl resulting from chloride exchange has been reported tomodify the structure and reduce the thermal stability of some peptides(Biochemistry, 5th edition, Berg et al. eds, Freeman; 2002); J Pept Sci.2007 Jan;13(1):37-43). TAT-NR2B9c is already a highly soluble peptide asan acetate salt. Accordingly, replacement of trifluoroacetate or acetatesalt with chloride would have appeared to have the disadvantages ofdecreased yield and possible reduced stability without any compensatingbenefit.

Surprisingly it has been found that a chloride salt of TAT-NR2B9cconfers significantly greater stability in the lyophilized form than anacetate salt of TAT-NR2B9c in the same formulation, or an acetate saltof TAT-NR2B9c lyophilized from saline. The chloride salt can remainsufficiently stable for clinical use even with storage at summer timeambient temperatures reaching or even exceeding 37° C. for severalyears. The greater stability of the chloride salt over acetate more thancompensates for any greater effort or reduced yield required inreplacing trifluoroacetate as a salt. The present invention provideslyophilized formulations of active agents, particularly of TAT-NR2B9c asa chloride salt. Such formulations are stable at ambient temperature(e.g., at least 20° C.) thus facilitating maintenance of supplies ofsuch a formulation in ambulances or the like or with emergency personnelfor administration at the scene of illness or accident or between suchscene and a medical facility.

Lyophilized formulations are prepared from a prelyophilized formulationcomprising an active agent, a buffer, a bulking agent and water. Othercomponents, such as cryo or lyopreservatives, a tonicity agentpharmaceutically acceptable carriers and the like may or may not bepresent. A preferred active agent is a chloride salt of TAT-NR2B9c. Apreferred buffer is histidine. A preferred bulking agent is trehalose.Trehalose also serves as a cryo and lyo-preservative. An exemplaryprelyophilized formulation comprises the active agent (e.g., chloridesalt of TAT-NR2B9c), histidine (10-100 mM, 15-100 mM 15-80 mM, 40-60 mMor 15-60 mM, for example, 20 mM or optionally 50 mM, or 20-50 mM)) andtrehalose (50-200 mM, preferably 80-160 mM, 100-140 mM, more preferably120 mM). The pH is 5.5 to 7.5, more preferably, 6-7, more preferably6.5. The concentration of active agent (e.g., chloride salt ofTAT-NR2B9c) is 20-200 mg/ml, preferably 50-150 mg/ml, more preferably70-120 mg/ml or 90 mg/ml. Thus, an exemplary prelyophilized formulationis 20 mM histidine, 120 mM trehalose, and 90 mg/ml chloride salt ofTAT-NR2B9c. Optionally an acetylation scavenger, such as lysine can beincluded, as described in co-pending application US20170112769, tofurther reduce any residual acetate or trifluoroacetate in theformulation.

After lyophilization, lyophilized formulations have a low-water content,preferably from about 0%-5% water, more preferably below 2.5% water byweight. Lyophilized formulations can be stored in a freezer (e.g., −20or −70° C.), in a refrigerator (0-4° C.) or at room temperature (20-25°C.).

Active agents are reconstituted in an aqueous solution, preferably waterfor injection or optionally normal saline (0.8-1.0% saline andpreferably 0.9% saline). Reconstitution can be to the same or a smalleror larger volume than the prelyophilized formulation. Preferably, thevolume is larger post-reconstitution than before (e.g., 3-6 timeslarger). For example, a prelyophilization volume of 3-5 ml can bereconstituted as a volume of 10 mL, 12 mL, 13.5 ml, 15 mL or 20 mL or10-20 mL among others. After reconstitution, the concentration ofhistidine is preferably 2-20 mM, e.g., 2-7 mM, 4.0-6.5 mM, 4.5 mM or 6mM; the concentration of trehalose is preferably 15-45 mM or 20-40 mM or25-27 mM or 35-37 mM. The concentration of lysine is preferably 100-300mM, e.g., 150-250 mM, 150-170 mM or 210-220 mM. The active agent ispreferably at a concentration of 10-30 mg/ml, for example 15-30, 18-20,20 mg/ml of active agent (e.g., TAT-NR2B9c) or 25-30, 26-28 or 27 mg/mLactive agent. An exemplary formulation after reconstitution has 4-5 mMhistidine, 26-27 mM trehalose, 150-170 mM lysine and 20 mg/ml TAT-NR2B9c(with concentrations rounded to the nearest integer). A second exemplaryformulation after reconstitution has 5-7 mM histidine, 35-37 mMtrehalose, 210-220 mM lysine and 26-28 mg/ml TAT-NR2B9c (withconcentrations rounded to the nearest integer). The reconstitutedformulation can be further diluted before administration such as byadding into a fluid bag containing normal saline for intravenousinfusion.

Any description of a formulation as comprising or including (or similarterminology) specified components should be understood as alternativelyor additional describing a formulation consisting of or consistingessentially of those specified components.

Methods of freeze drying are set forth, for example, in Methods inEnzymology, Vol. 22, Pages 33-39, Academic Press, New York (1971); andin Freeze-Drying, E. W. Flosdorf, Rheinhold, New York (1949). TAT-NR2B9cis preferably lyophilized in the same vial as that in which it will bereconstituted for use. An aqueous solution of TAT-NR2B9c is added to thevial optionally after filtering through a sterilizing filtration system,such as a 0.22 micron filter standardly used for peptides. Formulationscan be lyophilized in a controlled cycle, such as described in theExamples. A prelyophilized formulation can be placed in a vial, andlyophilized at reduced temperature and pressure. After lyophilization,vials can be sealed. For use, the lyophilizate is reconstituted withwater for injection, normal saline or other pharmaceutically acceptablecarrier or diluent.

A variety of containers are suitable for lyophilization. A containershould be able to withstand the outside pressure when the container issealed and stored under partial vacuum. The container should be made ofa material that allows a reasonable transfer of heat from outside toinside. The size of the container should be such that the solution to belyophilized occupies not more than 20% of the useful volume or may beoverfilled with an excess, in accord with then-prevailing USPrecommendations for the volume in a container. For example, a 0.5 mlsolution may be filled in a 3 ml vial. The vials may be made of glasse.g. borosilicate, or plastic, e.g. polypropylene.

Glass bottles commonly used for lyophilizing biological materials can beused. Another suitable container is a two-compartment syringe whereinone compartment contains the lyophilized TAT-NR2B9c peptide cake and theother compartment contains the aqueous diluent. After lyophilization iscomplete, the vacuum within the vials or ampules may be released byfilling the system with an inert gas, stoppered in place using standardequipment and then crimp sealed. Such a method will ensure a sterilefinal product. Other two-part solutions such as a bag with a breakableseal between the lyophilized drug compartment and the diluent can beused as well.

II. Active Agents

Although much of the description refers to the active agent TAT-NR2B9cfor purposes of exemplification, other active agents as described belowcan be prepared as chloride salts or formulated according to theprinciples described for TAT-NR2B9c. Specific concentrations given forTAT-NR2B9c can be used as is for other agents or converted to giveequimolar concentrations of the other agent and TAT-NR2B9c.

Active agents inhibit interaction between PSD-95 and one or more NMDARs(e.g., 2A, 2B, 2C or 2D) or nNOS (e.g., Swiss-Prot P29475) by binding toPSD-95. Such agents are useful for reducing one or more damaging effectsof stroke and other neurological conditions mediated at least in part byNMDAR excitotoxicity. Such agents include peptides having an amino acidsequence including or based on the PL motif of a NMDA Receptor or PDZdomain of PSD-95. Such peptides can also inhibit interactions betweenPSD-95 and nNOS and other glutamate receptors (e.g., kainite receptorsor AMPA receptors), such as KV1.4 and GluR6. Preferred peptides inhibitinteraction between PDZ domains 1 and 2 of postsynaptic density-95protein (PSD-95)(human amino acid sequence provided by Stathakism,Genomics 44(1):71-82 (1997)) and the C-terminal PL sequence of one ormore NMDA Receptor 2 subunits including the NR2B subunit of the neuronalN-methyl-D-aspartate receptor (Mandich et al., Genomics 22, 216-8(1994)). NMDAR2B has GenBank ID 4099612, a C-terminal 20 amino acidsFNGSSNGHVYEKLSSIESDV (SEQ ID NO:11) and a PL motif ESDV (SEQ ID NO:12).Preferred peptides inhibit the human forms of PSD-95 and human NMDARreceptors. However, inhibition can also be shown from species variantsof the proteins. A list of NMDA and glutamate receptors that can be usedappears below:

NMDA Receptors With PL Sequences C-terminal C-terminal internal PL NameGI or Acc# 20mer sequence 4mer sequence PL? ID NMDAR1 307302HPTDITGPLNLSDPSVST STVV X AA216 VV (SEQ ID NO: 13) (SEQ ID NO: 27)NMDAR1-1 292282 HPTDITGPLNLSDPSVST STVV X AA216 VV (SEQ ID NO: 13)(SEQ ID NO: 27) NMDAR1-4 472845 HPTDITGPLNLSDPSVST STVV X AA216VV (SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1- 2343286 HPTDITGPLNLSDPSVSTSTVV X AA216 3b VV (SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1- 2343288HPTDITGPLNLSDPSVST STVV X AA216 4b VV (SEQ ID NO: 13) (SEQ ID NO: 27)NMDAR1-2 11038634 RRAIEREEGQLQLCSRH HRES RES (SEQ ID NO: 14) (SEQ IDNO: 28) NMDAR1-3 11038636 RRAIEREEGQLQLCSRH HRES RES (SEQ ID NO: 14)(SEQ ID NO: 28) NMDAR2C 6006004 TQGFPGPCTWRRISSLES ESEV X AA180EV (SEQ ID NO: 15) (SEQ ID NO: 29) NMDAR3 560546 FNGSSNGHVYEKLSSIES ESDVX AA34.1 DV (SEQ ID NO: 11) (SEQ ID NO: 12) NMDAR3A 17530176AVSRKTELEEYQRTSRT TCES CES (SEQ ID NO: 16) (SEQ ID NO: 30) NMDAR2B4099612 FNGSSNGHVYEKLSSIES ESDV X DV (SEQ ID NO: 11) (SEQ ID NO: 12)NMDAR2A 558748 LNSCSNRRVYKKMPSIE ESDV X AA34.2 SDV (SEQ ID NO: 17)(SEQ ID NO: 12) NMDAR2D 4504130 GGDLGTRRGSAHFSSLE ESEV XSEV (SEQ ID NO: 18) (SEQ ID NO: 29) Glutamate AF009014 QPTPTLGLNLGNDPDRGGTSI (SEQ X receptor TSI (SEQ ID NO: 19) ID NO: 31) delta 2 GlutamateI28953 MQSIPCMSHSSGMPLGA ATGL (SEQ X receptor 1 TGL (SEQ ID NO: 20)ID NO: 32) Glutamate L20814 QNFATYKEGYNVYGIES SVKI (SEQ ID X receptor 2VKI (SEQ ID NO: 21) NO: 33) Glutamate AF167332 QNYATYREGYNVYGTESVKI (SEQ ID X receptor 3 SVKI (SEQ ID NO: 22) NO: 33) Glutamate U16129HTGTAIRQSSGLAVIASD SDLP (SEQ ID receptor 4 LP (SEQ ID NO: 23) NO: 34)Glutamate U16125 SFTSILTCHQRRTQRKET ETVA (SEQ X receptor 5VA (SEQ ID NO: 24) ID NO: 35) Glutamate U16126 EVINMHTFNDRRLPGKEETMA (SEQ X receptor 6 TMA (SEQ ID NO: 25) ID NO: 36)

Peptides can include or be based on a PL motif from the C-terminus ofany of the above subunits and have an amino acid sequence comprising[S/T]-X-[V/L]. This sequence preferably occurs at the C-terminus of thepeptides of the invention. Preferred peptides have an amino acidsequence comprising [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID NO:38) attheir C-terminus. Exemplary peptides comprise: ESDV (SEQ ID NO:12), ESEV(SEQ ID NO:29), ETDV (SEQ ID NO:39), E (SEQ ID NO:40), DTDV (SEQ IDNO:41), and D (SEQ ID NO:42) as the C-terminal amino acids. Twoparticularly preferred peptides are KLSSIESDV (SEQ ID NO:5), andKLSSIETDV (SEQ ID NO:43). Such peptides usually have 3-25 amino acids(without an internalization peptide), peptide lengths of 5-10 aminoacids, and particularly 9 amino acids (also without an internalizationpeptide) are preferred. In some such peptides, all amino acids are fromthe C-terminus of an NMDA receptor (not including amino acids from aninternalization peptide).

Peptides and peptidomimetics of the invention can contain modified aminoacid residues for example, residues that are N-alkylated. N-terminalalkyl modifications can include e.g., N-Methyl, N-Ethyl, N-Propyl,N-Butyl, N-Cyclohexylmethyl, N-Cyclyhexylethyl, N-Benzyl, N-Phenylethyl,N-phenylpropyl, N-(3, 4-Dichlorophenyl)propyl,N-(3,4-Difluorophenyl)propyl, and N-(Naphthalene-2-yl)ethyl).

Bach, J. Med. Chem. 51, 6450-6459 (2008) and WO 2010/004003 havedescribed a series of analogs of NR2B9c (SEQ ID NO:6). PDZ-bindingactivity is exhibited by peptides having only three C-terminal aminoacids (SDV). Bach also reports analogs having an amino acid sequencecomprising or consisting of X₁tSX₂V (SEQ ID NO:68), wherein t and S arealternative amino acids, X₁ is selected from among E, Q, and A, or ananalogue thereof, X₂ is selected from among A, Q, D, N, N-Me-A, N-Me-Q,N-Me-D, and N-Me-N or an analog thereof. Optionally the peptide isN-alkylated in the P3 position (third amino acid from C-terminus, i.e.position occupied by tS). The peptide can be N-alkylated with acyclohexane or aromatic substituent, and further comprises a spacergroup between the sub stituent and the terminal amino group of thepeptide or peptide analogue, wherein the spacer is an alkyl group,preferably selected from among methylene, ethylene, propylene andbutylene. The aromatic sub stituent can be a naphthalen-2-yl moiety oran aromatic ring substituted with one or two halogen and/or alkyl group.

Other modifications can also be incorporated without adversely affectingthe activity and these include substitution of one or more of the aminoacids in the natural L-isomeric form with amino acids in the D-isomericform. Thus, any amino acid naturally occurring in the L-configuration(which can also be referred to as the R or S, depending upon thestructure of the chemical entity) can be replaced with the amino acid ofthe same chemical structural type or a peptidomimetic, but of theopposite chirality, generally referred to as the D-amino acid, but whichcan additionally be referred to as the R- or S-form. Thus, apeptidomimetic may include 1, 2, 3, 4, 5, at least 50%, or all D-aminoacid resides. A peptidomimetic containing some or all D residues issometimes referred to an “inverso” peptide.

Peptidomimetics also include retro peptides. A retro peptide has areverse amino acid sequence. Peptidomimetics also include retro inversopeptides in which the order of amino acids is reversed from so theoriginally C-terminal amino acid appears at the N-terminus and D-aminoacids are used in place of L-amino acids. WO 2008/014917 describes aretro-inverso analog of Tat-NR2B9c having the amino acid sequencevdseisslk-rrrqrrldcrgyin (SEQ ID NO:69) (lower case letters indicating Damino acids), and reports it to be effective inhibiting cerebralischemia. Another effective peptide described herein is Rv-Tat-NR2B9c(RRRQRRKKRGYKLSSIESDV; SEQ ID NO:70).

A linker, e.g., a polyethylene glycol linker, can be used to dimerizethe active moiety of the peptide or the peptidomimetic to enhance itsaffinity and selectivity towards proteins containing tandem PDZ domains.See e.g., Bach et al., (2009) Angew. Chem. Int. Ed. 48:9685-9689 and WO2010/004003. A PL motif-containing peptide is preferably dimerized viajoining the N-termini of two such molecules, leaving the C-termini free.Bach further reports that a pentamer peptide IESDV (SEQ ID NO:71) fromthe C-terminus of NMDAR 2B was effective in inhibiting binding of NMDAR2B to PSD-95. IETDV (SEQ ID NO:73) can also be used instead of IESDV(SEQ ID NO:71). Optionally, about 2-10 copies of a PEG can be joined intandem as a linker. Optionally, the linker can also be attached to aninternalization peptide or lipidated to enhance cellular uptake.Examples of illustrative dimeric inhibitors are shown below (see Bach etal., PNAS 109 (2012) 3317-3322). Any of the PSD-95 inhibitors disclosedherein can be used instead of IETDV (SEQ ID NO:71), and anyinternalization peptide or lipidating moiety can be used instead of tat.Other linkers to that shown can also be used.

IETAV is assigned SEQ ID NO:26, YGRKKRRQRRR SEQ ID NO:2, and rrrqrrkkr,SEQ ID NO:10, lower case letters indicated D-amino acids.

Appropriate pharmacological activity of peptides, peptidomimetics orother agent can be confirmed if desired, using previously described ratmodels of stroke before testing in the primate and clinical trialsdescribed in the present application. Peptides or peptidomimetics canalso be screened for capacity to inhibit interactions between PSD-95 andNMDAR 2B using assays described in e.g., US 20050059597, which isincorporated by reference. Useful peptides typically have IC50 values ofless than 50 μM, 25 μM, 10 μM, 0.1 μM or 0.01 μM in such an assay.Preferred peptides typically have an IC50 value of between 0.001-1 μM,and more preferably 0.001-0.05, 0.05-0.5 or 0.05 to 0.1 μM. When apeptide or other agent is characterized as inhibiting binding of oneinteraction, e.g., PSD-95 interaction to NMDAR2B, such description doesnot exclude that the peptide or agent also inhibits another interaction,for example, inhibition of PSD-95 binding to nNOS.

Peptides such as those just described can optionally be derivatized(e.g., acetylated, phosphorylated, myristoylated, geranylated, pegylatedand/or glycosylated) to improve the binding affinity of the inhibitor,to improve the ability of the inhibitor to be transported across a cellmembrane or to improve stability. As a specific example, for inhibitorsin which the third residue from the C-terminus is S or T, this residuecan be phosphorylated before use of the peptide.

A pharmacological agent can be linked to an internalization peptide tofacilitate uptake into cells and/or across the blood brain barrier.Internalization peptides are a well-known class of relatively shortpeptides that allow many cellular or viral proteins to traversemembranes. Internalization peptides, also known as cell membranetransduction peptides or cell penetrating peptides can have e.g., 5-30amino acids. Such peptides typically have a cationic charge from anabove normal representation (relative to proteins in general) ofarginine and/or lysine residues that is believed to facilitate theirpassage across membranes. Some such peptides have at least 5, 6, 7 or 8arginine and/or lysine residues. Examples include the antennapediaprotein (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (and variantsthereof), the tat protein of human immunodeficiency virus, the proteinVP22, the product of the UL49 gene of herpes simplex virus type 1,Penetratin, SynB1 and 3, Transportan, Amphipathic, gp41NLS, polyArg, andseveral plant and bacterial protein toxins, such as ricin, abrin,modeccin, diphtheria toxin, cholera toxin, anthrax toxin, heat labiletoxins, and Pseudomonas aeruginosa exotoxin A (ETA). Other examples aredescribed in the following references (Temsamani, Drug Discovery Today,9(23):1012-1019, 2004; De Coupade, Biochem J., 390:407-418, 2005; SaalikBioconjugate Chem. 15: 1246-1253, 2004; Zhao, Medicinal Research Reviews24(1):1-12, 2004; Deshayes, Cellular and Molecular Life Sciences62:1839-49, 2005); Gao, ACS Chem. Biol. 2011, 6, 484-491, SG3(RLSGMNEVLSFRWL (SEQ ID NO:9)), Stalmans PLoS ONE 2013, 8(8) e71752,1-11 and supplement; Figueiredo et al., IUBMB Life 66, 182-194 (2014);Copolovici et al., ACS Nano, 8, 1972-94 (2014); Lukanowski Biotech J. 8,918-930 (2013); Stockwell, Chem. Biol. Drug Des. 83, 507-520 (2014);Stanzl et al. Accounts. Chem. Res/ 46, 2944-2954 (2013); (allincorporated by reference).

A preferred internalization peptide is tat from the HIV virus. A tatpeptide reported in previous work comprises or consists of the standardamino acid sequence YGRKKRRQRRR (SEQ ID NO:2) found in HIV Tat protein.If additional residues flanking such a tat motif are present (beside thepharmacological agent) the residues can be for example natural aminoacids flanking this segment from a tat protein, spacer or linker aminoacids of a kind typically used to join two peptide domains, e.g., gly(ser)4 (SEQ ID NO:44), TGEKP (SEQ ID NO:45), GGRRGGGS (SEQ ID NO:46), orLRQRDGERP (SEQ ID NO:47) (see, e.g., Tang et al. (1996), J. Biol. Chem.271, 15682-15686; Hennecke et al. (1998), Protein Eng. 11, 405-410)), orcan be any other amino acids that do not significantly reduce capacityto confer uptake of the variant without the flanking residues.Preferably, the number of flanking amino acids other than an activepeptide does not exceed ten on either side of YGRKKRRQRRR (SEQ ID NO:2).One suitable tat peptide comprising additional amino acid residuesflanking the C-terminus of YGRKKRRQRRR (SEQ ID NO:2) is YGRKKRRQRRRPQ(SEQ ID NO:48). However, preferably, no flanking amino acids arepresent. Other tat peptides that can be used include GRKKRRQRRRPQ (SEQID NO:4) and GRKKRRQRRRP (SEQ ID NO:72).

Variants of the above tat peptide having reduced capacity to bind toN-type calcium channels are described by WO/2008/109010. Such variantscan comprise or consist of an amino acid sequence XGRKKRRQRRR (SEQ IDNO:49), in which X is an amino acid other than Y or nothing (in whichcase G is a free N-terminal residue). A preferred tat peptide has theN-terminal terminal Y residue substituted with F. Thus, a tat peptidecomprising or consisting of FGRKKRRQRRR (SEQ ID NO:3) is preferred.Another preferred variant tat peptide consists of GRKKRRQRRR (SEQ IDNO:1). Another preferred tat peptide comprises or consists of RRRQRRKKRGor RRRQRRKKRGY (amino acidsl-10 or 1-11 of SEQ ID NO:70). Other tatderived peptides that facilitate uptake of a pharmacological agentwithout inhibiting N-type calcium channels include those presentedbelow.

X-FGRKKRRQRRR (F-Tat) (SEQ ID NO: 8) X-GKKKKKQKKK (SEQ ID NO: 50)X-RKKRRQRRR (SEQ ID NO: 51) X-GAKKRRQRRR (SEQ ID NO: 52)X-AKKRRQRRR (SEQ ID NO: 53) X-GRKARRQRRR (SEQ ID NO: 54)X-RKARRQRRR (SEQ ID NO: 55) X-GRKKARQRRR (SEQ ID NO: 56)X-RKKARQRRR (SEQ ID NO: 57) X-GRKKRRQARR (SEQ ID NO: 58)X-RKKRRQARR (SEQ ID NO: 59) X-GRKKRRQRAR (SEQ ID NO: 60)X-RKKRRQRAR (SEQ ID NO: 61) X-RRPRRPRRPRR (SEQ ID NO: 62)X-RRARRARRARR (SEQ ID NO: 63) X-RRRARRRARR (SEQ ID NO: 64)X-RRRPRRRPRR (SEQ ID NO: 65) X-RRPRRPRR (SEQ ID NO: 66)X-RRARRARR (SEQ ID NO: 67)

X can represent a free amino terminus, one or more amino acids, or aconjugated moiety. Internalization peptides can be used in inverso orretro or inverso retro form with or without the linked peptide orpeptidomimetic being in such form. For example, a preferred chimericpeptide has an amino acid sequence comprising or consisting ofYGRKKRRQRRR-KLSSIESDV (SEQ ID NO:6, also known as TAT-NR2B9c orTat-NR2B9c), or YGRKKRRQRRR-KLSSIETDV (SEQ ID NO:7). Other preferredchimeric peptides differ from SEQ ID NO:6 or NO:7 by up to 1, 2, 3, 4 or5 amino acid substitutions, deletions or additions (internal or at theends). Other preferred peptides include RRRQRRKKRGY-KLSSIESDV (SEQ IDNO:70, also known as RvTat-NR2B9c or having an amino acid sequencecomprising or consisting of RRRQRRKKRGY-KLSSIETDV (SEQ ID NO:37).

Internalization peptides can be attached to pharmacological agents byconventional methods. For example, the agents can be joined tointernalization peptides by chemical linkage, for instance via acoupling or conjugating agent. Numerous such agents are commerciallyavailable and are reviewed by S. S. Wong, Chemistry of ProteinConjugation and Cross-Linking, CRC Press (1991). Some examples ofcross-linking reagents include J-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N,N′-(1,3-phenylene) bismaleimide;N,N′-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11carbon methylene bridges (which relatively specific for sulfhydrylgroups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversiblelinkages with amino and tyrosine groups). Other cross-linking reagentsinclude p,p′-difluoro-m, m′-dinitrodiphenylsulfone (which formsirreversible cross-linkages with amino and phenolic groups); dimethyladipimidate (which is specific for amino groups);phenol-1,4-disulfonylchloride (which reacts principally with aminogroups); hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate (which reacts principally with amino groups);glutaraldehyde (which reacts with several different side chains) anddisdiazobenzidine (which reacts primarily with tyrosine and histidine).

For pharmacological agents that are peptides attachment to aninternalization peptide can be achieved by generating a fusion proteincomprising the peptide sequence fused, preferably at its N-terminus, toan internalization peptide.

Instead of or as well as linking a peptide (or other agent) inhibitingPSD-95 to an internalization peptide, such a peptide can be linked to alipid (lipidation) to increase hydrophobicity of the conjugate relativeto the peptide alone and thereby facilitate passage of the linkedpeptide across cell membranes and/or across the brain barrier.Lipidation is preferably performed on the N-terminal amino acid but canalso be performed on internal amino acids, provided the ability of thepeptide to inhibit interaction between PSD-95 and NMDAR 2B is notreduced by more than 50%. Preferably, lipidation is performed on anamino acid other than one of the four most C-terminal amino acids.Lipids are organic molecules more soluble in ether than water andinclude fatty acids, glycerides and sterols. Suitable forms oflipidation include myristoylation, palmitoylation or attachment of otherfatty acids preferably with a chain length of 10-20 carbons, such aslauric acid and stearic acid, as well as geranylation,geranylgeranylation, and isoprenylation. Lipidations of a type occurringin posttranslational modification of natural proteins are preferred.Lipidation with a fatty acid via formation of an amide bond to thealpha-amino group of the N-terminal amino acid of the peptide is alsopreferred. Lipidation can be by peptide synthesis including aprelipidated amino acid, be performed enzymatically in vitro or byrecombinant expression, by chemical crosslinking or chemicalderivatization of the peptide. Amino acids modified by myristoylationand other lipid modifications are commercially available.

Lipidation preferably facilitates passage of a linked peptide (e.g.,KLSSIESDV (SEQ ID NO:5), or KLSSIETDV (SEQ ID NO:43)) across a cellmembrane and/or the blood brain barrier without causing a transientreduction of blood pressure as has been found when a standard tatpeptide is administered at high dosage (e.g., at or greater than 3mg/kg), or at least with smaller reduction that than the same peptidelinked to a standard tat peptide.

Pharmacologic peptides, optionally fused to tat peptides, can besynthesized by solid phase synthesis or recombinant methods.Peptidomimetics can be synthesized using a variety of procedures andmethodologies described in the scientific and patent literature, e.g.,Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley &Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby(1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.

III. Salts

Peptides of the type described above are typically made by solid statesynthesis. Because solid state synthesis uses trifluoroacetate (TFA) toremove protecting groups or remove peptides from a resin, peptides aretypically initially produced as trifloroacetate salts. Thetrifluoroacetate can be replaced with another anion by for example,binding the peptide to a solid support, such as a column, washing thecolumn to remove the existing counterion, equilibrating the column witha solution containing the new counterion and then eluting the peptide,e.g., by introducing a hydrophobic solvent such as acetonitrile into thecolumn. Replacement of trifluoroacetate with acetate can be done with anacetate wash as the last step before peptide is eluted in an otherwiseconventional solid state synthesis. Replacing trifluoroacetate oracetate with chloride can be done with a wash with ammonium chloridefollowed by elution. Use of a hydrophobic support is preferred andpreparative reverse phase HPLC is particularly preferred for the ionexchange. Trifluoroacetate can be replaced with chloride directly or canfirst be replaced by acetate and then the acetate replaced by chloride.

Counterions, whether trifluoroacetate, acetate or chloride, bind topositively charged atoms on TAT-NR2B9c, particularly the N-terminalamino group and amino side chains arginine and lysine residues. Althoughpractice of the invention, it is not dependent on understanding theexact stochiometery of peptide to anion in a salt of TAT-NR2B9c, it isbelieved that up to about 9 counterion molecules are present permolecule of salt.

Although replacement of one counterion by another takes placeefficiently, the purity of the final counterion may be less than 100%.Thus, reference to a chloride salt of TAT-NR2B9c or other active agentmeans that in a preparation of the salt, chloride is the predominantanion by weight (or moles) over all other anions present in theaggregate in the salt. In other words, chloride constitutes greater than50% and preferably greater than 75%, 95%, 99%, 99.5% or 99.9% by weightor moles of the all anions present in the salt. In such a salt orformulation prepared from the salt, acetate and trifluoroacetate incombination and individually constitutes less than 50%, 25%, 5%, 5% 0.5%or 0.1 of the anions in the salt or formulation.

IV. Diseases

The lyophilized formulations are useful in treating a variety ofdiseases, particularly neurological diseases, and especially diseasesmediated in part by excitotoxity. Such diseases and conditions includestroke, epilepsy, hypoxia, subarachnoid hemorrhage, traumatic injury tothe CNS not associated with stroke such as traumatic brain injury andspinal cord injury, other cerebral ischemia, Alzheimer's disease andParkinson's disease. Other neurological diseases treatable by agents ofthe invention not known to be associated with excitotoxicity includeanxiety and pain.

A stroke is a condition resulting from impaired blood flow in the CNSregardless of cause. Potential causes include embolism, hemorrhage andthrombosis. Some neuronal cells die immediately as a result of impairedblood flow. These cells release their component molecules includingglutamate, which in turn activates NMDA receptors, which raiseintracellular calcium levels, and intracellular enzyme levels leading tofurther neuronal cell death (the excitotoxicity cascade). The death ofCNS tissue is referred to as infarction. Infarction Volume (i.e., thevolume of dead neuronal cells resulting from stroke in the brain) can beused as an indicator of the extent of pathological damage resulting fromstroke. The symptomatic effect depends both on the volume of aninfarction and where in the brain it is located. Disability index can beused as a measure of symptomatic damage, such as the Rankin StrokeOutcome Scale (Rankin, Scott Med J;2:200-15 (1957)) and the BarthelIndex. The Rankin Scale is based on assessing directly the globalconditions of a patient as follows.

0: No symptoms at all

1: No significant disability despite symptoms; able to carry out allusual duties and activities.

2: Slight disability; unable to carry out all previous activities butable to look after own affairs without assistance.

3: Moderate disability requiring some help, but able to walk withoutassistance

4: Moderate to severe disability; unable to walk without assistance andunable to attend to own bodily needs without assistance.

5: Severe disability; bedridden, incontinent, and requiring constantnursing care and attention.

The Barthel Index is based on a series of questions about the patient'sability to carry out 10 basic activities of daily living resulting in ascore between 0 and 100, a lower score indicating more disability(Mahoney et al, Maryland State Medical Journal 14:56-61 (1965)).

Alternatively stroke severity/outcomes can be measured using the NIHstroke scale, available at world wide web ninds.nih.gov/doctors/NIHStroke ScaleJBooklet.pdf.

The scale is based on the ability of a patient to carry out 11 groups offunctions that include assessments of the patient's level ofconsciousness, motor, sensory and language functions.

An ischemic stroke refers more specifically to a type of stroke thatcaused by blockage of blood flow to the brain. The underlying conditionfor this type of blockage is most commonly the development of fattydeposits lining the vessel walls. This condition is calledatherosclerosis. These fatty deposits can cause two types ofobstruction. Cerebral thrombosis refers to a thrombus (blood clot) thatdevelops at the clogged part of the vessel “Cerebral embolism” refersgenerally to a blood clot that forms at another location in thecirculatory system, usually the heart and large arteries of the upperchest and neck. A portion of the blood clot then breaks loose, entersthe bloodstream and travels through the brain's blood vessels until itreaches vessels too small to let it pass. A second important cause ofembolism is an irregular heartbeat, known as arterial fibrillation. Itcreates conditions in which clots can form in the heart, dislodge andtravel to the brain. Additional potential causes of ischemic stroke arehemorrhage, thrombosis, dissection of an artery or vein, a cardiacarrest, shock of any cause including hemorrhage, and iatrogenic causessuch as direct surgical injury to brain blood vessels or vessels leadingto the brain or cardiac surgery. Ischemic stroke accounts for about 83percent of all cases of stroke.

Transient ischemic attacks (TIAs) are minor or warning strokes. In aTIA, conditions indicative of an ischemic stroke are present and thetypical stroke warning signs develop. However, the obstruction (bloodclot) occurs for a short time and tends to resolve itself through normalmechanisms. Patients undergoing heart surgery are at particular risk oftransient cerebral ischemic attack.

Hemorrhagic stroke accounts for about 17 percent of stroke cases. Itresults from a weakened vessel that ruptures and bleeds into thesurrounding brain. The blood accumulates and compresses the surroundingbrain tissue. The two general types of hemorrhagic strokes areintracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic strokeresult from rupture of a weakened blood vessel ruptures. Potentialcauses of rupture from a weakened blood vessel include a hypertensivehemorrhage, in which high blood pressure causes a rupture of a bloodvessel, or another underlying cause of weakened blood vessels such as aruptured brain vascular malformation including a brain aneurysm,arteriovenous malformation (AVM) or cavernous malformation. Hemorrhagicstrokes can also arise from a hemorrhagic transformation of an ischemicstroke which weakens the blood vessels in the infarct, or a hemorrhagefrom primary or metastatic tumors in the CNS which contain abnormallyweak blood vessels. Hemorrhagic stroke can also arise from iatrogeniccauses such as direct surgical injury to a brain blood vessel. Ananeurysm is a ballooning of a weakened region of a blood vessel. If leftuntreated, the aneurysm continues to weaken until it ruptures and bleedsinto the brain. An arteriovenous malformation (AVM) is a cluster ofabnormally formed blood vessels. A cavernous malformation is a venousabnormality that can cause a hemorrhage from weakened venous structures.Any one of these vessels can rupture, also causing bleeding into thebrain. Hemorrhagic stroke can also result from physical trauma.Hemorrhagic stroke in one part of the brain can lead to ischemic strokein another through shortage of blood lost in the hemorrhagic stroke.

One patient class amenable to treatments are patients undergoing asurgical procedure that involves or may involve a blood vessel supplyingthe brain, or otherwise on the brain or CNS. Some examples are patientsundergoing cardiopulmonary bypass, carotid stenting, diagnosticangiography of the brain or coronary arteries of the aortic arch,vascular surgical procedures and neurosurgical procedures. Additionalexamples of such patients are discussed in section IV above. Patientswith a brain aneurysm are particularly suitable. Such patients can betreated by a variety of surgical procedures including clipping theaneurysm to shut off blood, or performing endovascular surgery to blockthe aneurysm with small coils or introduce a stent into a blood vesselfrom which an aneurysm emerges, or inserting a microcatheter.Endovascular procedures are less invasive than clipping an aneurysm andare associated with a better patient outcome but the outcome stillincludes a high incidence of small infarctions. Such patients can betreated with an inhibitor of PSD95 interaction with NMDAR 2B andparticularly the agents described above including the peptideYGRKKRRQRRRKLSSIESDV (SEQ ID NO:6, also known as Tat-NR2B9c). The timingof administration relative to performing surgery can be as describedabove for the clinical trial.

Another class of patients amenable to treatment are patients having asubarachnoid hemorrhage with or without an aneurysm (see US 61/570,264).

IV. Effective Regimes of Administration

After reconstitution, a lyophilized formulation, is administered suchthat the active agent (e.g., NR2B9c) is administered in an amount,frequency and route of administration effective to cure, reduce orinhibit further deterioration of at least one sign or symptom of adisease in a patient having the disease being treated. A therapeuticallyeffective amount means an amount of active agent sufficientsignificantly to cure, reduce or inhibit further deterioration of atleast one sign or symptom of the disease or condition to be treated in apopulation of patients (or animal models) suffering from the diseasetreated with an agent of the invention relative to the damage in acontrol population of patients (or animal models) suffering from thatdisease or condition who are not treated with the agent. The amount isalso considered therapeutically effective if an individual treatedpatient achieves an outcome more favorable than the mean outcome in acontrol population of comparable patients not treated by methods of theinvention. A therapeutically effective regime involves theadministration of a therapeutically effective dose at a frequency androute of administration needed to achieve the intended purpose.

For a patient suffering from stroke or other ischemic condition, theactive agent is administered in a regime comprising an amount frequencyand route of administration effective to reduce the damaging effects ofstroke or other ischemic condition. When the condition requiringtreatment is stroke, the outcome can be determined by infarction volumeor disability index, and a dosage is considered therapeuticallyeffective if an individual treated patient shows a disability of two orless on the Rankin scale and 75 or more on the Barthel scale, or if apopulation of treated patients shows a significantly improved (i.e.,less disability) distribution of scores on a disability scale than acomparable untreated population, see Lees et at L, N Engl J Med2006;354:588-600. A single dose of agent is usually sufficient fortreatment of stroke.

The invention also provides methods and formulations for the prophylaxisof a disorder in a subject at risk of that disorder. Usually such asubject has an increased likelihood of developing the disorder (e.g., acondition, illness, disorder or disease) relative to a controlpopulation. The control population for instance can comprise one or moreindividuals selected at random from the general population (e.g.,matched by age, gender, race and/or ethnicity) who have not beendiagnosed or have a family history of the disorder. A subject can beconsidered at risk for a disorder if a “risk factor” associated withthat disorder is found to be associated with that subject. A risk factorcan include any activity, trait, event or property associated with agiven disorder, for example, through statistical or epidemiologicalstudies on a population of subjects. A subject can thus be classified asbeing at risk for a disorder even if studies identifying the underlyingrisk factors did not include the subject specifically. For example, asubject undergoing heart surgery is at risk of transient cerebralischemic attack because the frequency of transient cerebral ischemicattack is increased in a population of subjects who have undergone heartsurgery as compared to a population of subjects who have not.

Other common risk factors for stroke include age, family history,gender, prior incidence of stroke, transient ischemic attack or heartattack, high blood pressure, smoking, diabetes, carotid or other arterydisease, atrial fibrillation, other heart diseases such as heartdisease, heart failure, dilated cardiomyopathy, heart valve diseaseand/or congenital heart defects; high blood cholesterol, and diets highin saturated fat, trans fat or cholesterol.

In prophylaxis, a lyophilized formulation after reconstitution isadministered to a patient at risk of a disease but not yet having thedisease in an amount, frequency and route sufficient to prevent, delayor inhibit development of at least one sign or symptom of the disease. Aprophylactically effective amount means an amount of agent sufficientsignificantly to prevent, inhibit or delay at least one sign or symptomof the disease in a population of patients (or animal models) at risk ofthe disease relative treated with the agent compared to a controlpopulation of patients (or animal models) at risk of the disease nottreated with a chimeric agent of the invention. The amount is alsoconsidered prophylactically effective if an individual treated patientachieves an outcome more favorable than the mean outcome in a controlpopulation of comparable patients not treated by methods of theinvention. A prophylactically effective regime involves theadministration of a prophylactically effective dose at a frequency androute of administration needed to achieve the intended purpose. Forprophylaxis of stroke in a patient at imminent risk of stroke (e.g., apatient undergoing heart surgery), a single dose of agent is usuallysufficient.

Depending on the agent, administration can be parenteral, intravenous,nasal, oral, subcutaneous, intra-arterial, intracranial, intrathecal,intraperitoneal, topical, intranasal or intramuscular. Intravenousadministration is preferred for peptide agents.

For administration to humans, a preferred dose of active agent (e.g.,Tat-NR2B9c) is 2-3 mg/kg and more preferably 2.6 mg/kg. Indicateddosages should be understood as including the margin of error inherentin the accuracy with which dosages can be measured in a typical hospitalsetting. Such amounts are for single dose administration, i.e., one doseper episode of disease.

Active agents, such as Tat-NR2B9c are preferably delivered by infusioninto a blood vessel, more preferably by intravenous infusion. The timeof the infusion can affect both side effects (due e.g., to mast celldegranulation and histamine release) and efficacy. In general, for agiven dosage level, a shorter infusion time is more likely to lead tohistamine release. However, a shorter infusion time also may result inimproved efficacy. Although practice of the invention is not dependenton an understanding of mechanism, the latter result can be explainedboth because of the delay being significant relative to development ofpathology in the patient and because of the delay being significantrelative to the plasma half-life of the chimeric agent, as a result ofwhich the chimeric agent does not reach an optimal therapeutic level.For the chimeric agent Tat-NR2B9c, a preferred infusion time providing abalance between these considerations is 5-15 minutes and more preferably10 min. Indicated times should be understood as including a marking oferror of +/−10%. Infusion times do not include any extra time for a washout diffusion to wash out any remaining droplets from an initialdiffusion that has otherwise proceeded to completion. The infusion timesfor Tat-NR2B9c can also serve as a guide for other active agents.

Although the invention has been described in detail for purposes ofclarity of understanding, certain modifications may be practiced withinthe scope of the appended claims. All publications, accession numbers,and patent documents cited in this application are hereby incorporatedby reference in their entirety for all purposes to the same extent as ifeach were so individually denoted. To the extent more than one sequenceis associated with an accession number at different times, the sequencesassociated with the accession number as of the effective filing date ofthis application is meant. The effective filing date is the date of theearliest priority application disclosing the accession number inquestion. Unless otherwise apparent from the context any element,embodiment, step, feature or aspect of the invention can be performed incombination with any other.

EXAMPLES

Examples 1-7 show that an acetate salt of TAT-NR2B9c can be formulatedin lyophilized form with histidine and trehalose. Example 10 shows thatthe chloride salt of TAT-NR2B9c offers significantly greater stabilitythan the acetate salt in an otherwise identical lyophilized formulation.Example 9 shows that the chloride salt of TAT-NR2B9c formulated inhistidine and trehalose also gives improved stability relative to apreviously described lyophilized formulation of TAT-NR2B9c from normalsaline.

Example 1: Demonstration that Standard Buffers and Excipients Do NotInterfere With the Efficacy of TAT-NR2B9c in Vivo

Five liquid toxicology formulations were compounded targeted at a 20mg/mL concentration of TAT-NR2B9c. Table 1 includes the vehiclecomposition, Lot Number, and the potency, purity and pH at the time ofcompounding. Approximately 5 mL of each formulation was vialed fortesting. Vials were frozen at −20 to simulate transport or liquidstorage conditions.

TABLE 1 composition of TAT-NR2B9c formulations for efficacy testing invivo Potency¹ Purity² Formulation # Vehicle Composition PTek Lot #(mg/mL) (% Area of NA-1 peak) pH³ 1 50 mM sodium phosphate, 1205-1-17-120.5 97.87 6.7 76.9 mM NaCl, pH 7.0 2 50 mM sodium phosphate,1205-1-17-2 20.0 96.34² 6.5 154 mM Mannitol, pH 7.0 3 50 mM histidine,1205-1-17-3 19.9 98.38 6.4 154 mM Mannitol, pH 6.5 4 50 mM histidine,1205-1-17-4 20.8 99.16 6.4 154 mM Trehalose, pH 6.5 5 50 mM histidine,1205-1-18-1 19.4 98.81 6.4 5% Dextran-40, pH 6.5 ¹Potency and puritywere evaluated by RP-HPLC analysis using a TFA method ²The purity offormulation #2 is notably lower than the other formulations. ³The pH ofthe phosphate buffered formulations noticeably deviated from the initialbuffer pH of 7.0.

It was noted that phosphate buffered formulation did not maintain pH aswell as the histidine buffers did between formulation and testing,indicating that histidine may be a superior buffer for formulation.

Formulations 1-5 were tested in the 3-PIAL Vessel Occlusions (3PVO)model of stroke in rats. Rats subjected to stroke were given one of theformulations by intravenous administration into the femoral vein, andthen the animals were sacrificed 24 hours after the stroke. Brains wereharvested, fixed and stained with triphenyltetrazolium chloride (TTC) tovisualize the ischemic portions of the brain. All of the formulationstested were able to provide significant neuroprotection in animalsrelative to the saline-only control (FIG. 1).

Methods Three Pial Vessel Occlusion Model of Ischemia

Experiments were performed on rats. For permanent three pial vesselsocclusion (3PVO) was performed as described previously [Angiogenicprotection from focal ischemia with angiotensin II type 1 receptorblockade in the rat. Forder et al., Am J Physiol Heart Circ Physiol.2005 April;288(4):H1989-96]. In brief, 250 g to 350 g rats wereanesthetized with a 0.5 ml/kg intramuscular injection of ketamine (100mg/kg), acepromazine (2 mg/kg), and xylazine (5 mg/kg), supplementedwith one-third of the initial dose as required. An anal temperatureprobe was inserted, and the animal was placed on a heating padmaintained at about 37° C. The skull was exposed via a midline incisionand scraped free of tissue. Using a dissecting microscope and apneumatic dental drill, a 6- to 8-mm cranial window was made over theright somatosensory cortex (2 mm caudal and 5 mm lateral to bregma) bydrilling a rectangle through the skull and lifting off the piece ofskull while keeping the dura intact. The 3 pial arteriolar middlecerebral artery branches were cauterized around the barrel cortex wereselected and electrically cauterized through the dura. After thecauterizations, the scalp was sutured. Each rat was returned to itsindividual cage under a heating lamp to maintain body temperature untilthe rat fully recovered. Food and water was supplied. One hour after3PVO ischemia the rats were injected with NA1 formulations at 3 nmol/gin ˜0.45 mL saline based upon rat weight. Doses were administered over 5minutes.

Twenty-four hours after surgery, the brain was quickly harvested.Coronal slices (2 mm) were taken through the brain and incubated in 2%triphenyltetrazolium chloride (TTC) (Sigma-Aldrich St. Louis Mo.) for 15min at 37° C. Images were scanned (CanoScan 4200F Canon) andquantitated.

Example 2: Determination of TAT-NR2B9c Stability in Different Buffersand at Different pH Values Screening of Buffers

Ten buffers were compounded at 1 mg/mL TAT-NR2B9c for excipientscreening. Samples were stored at 25° C./60% relative humidity (RH) and40° C./75% RH. Samples were tested for stability (purity) at t=0 and t=1week for purity by RP-HPLC (TFA and MSA methods), and the results areshown in Tables 2 and 3.

Results indicate improved stability for TAT-NR2B9c in liquid mediabuffered between pH 6.0 and pH 6.5. Degradation appears to increaseoutside of this range in either direction. Data generated with the MSAmethod showed clear degradation patterns that were both pH and bufferingspecies dependent, and provided valuable insight into future formulationdevelopment. Results for main peak purity by % HPLC Area using the MSAmethod are provided in Table 2, while results for main peak purity by %HPLC Area using the TFA method are provided in Table 3.

TABLE 2 % Area of the Main Peak by MSA Method, TAT-NR2B9c Sample t = 0 t= 1 week 25° C. t = 1 week 40° C. His, 6.0 98.5 98.5 98.0 His, 6.5 98.598.6 97.3 His, 7.0 98.5 98.4 97.0 Phos, 6.0 98.5 98.2 97.0 Phos, 6.598.5 97.9 97.3 Phos, 7.0 98.5 97.9 96.0 Phos, 7.5 98.5 97.6 95.2 Citr,5.5 98.5 98.3 94.4 Citr, 6.0 98.5 98.4 97.4 Citr, 6.5 98.5 98.7 97.7

TABLE 3 % Area of the Main Peak by TFA Method, TAT-NR2B9c Sample t = 0 t= 1 week 25° C. t = 1 week 40° C. His, pH 6.0 99.5 98.8 99.2 His, pH 6.599.5 98.4 99.4 His, pH 7.0 99.5 98.3 96.7 Phos, 6.0 99.5 99.8 97.9 Phos,6.5 99.5 99.5 98.6 Phos, 7.0 99.5 98.6 98.3 Phos, 7.5 99.5 98.0 93.2Citr, 5.5 99.5 98.0 95.1 Citr, 6.0 99.5 99.0 98.2 Citr, 6.5 99.5 99.599.1

Results indicate that TAT-NR2B9c solution stability is best maintainedin pH 6.0 to 6.5 buffered media and the vehicle is still well toleratedfor administration by IV. In general, histidine and citrate bufferingsystems were able to maintain TAT-NR2B9c in an intact form even whenkept at accelerated stability conditions of 25° C. or 40° C. for 1 week.

There are several factors to consider when selecting a bufferingspecies: the specific degradation patterns that occur in each media, andany data on identified related substances or toxicology concerns maystreamline the decision process if specified related substances shouldbe avoided. For the period tested, histidine and citrate buffers betweenpH 6 and 6.5 showed few degradation products. The histidine bufferitself used in this study contained a contaminant that was present inthe histidine buffer in the absence of added TAT-NR2B9c. Therefore,identification of a supplier of histidine without such a contaminantwould make analysis simpler. Table 4 provides a summary of the bufferspecies from the standpoint of TAT-NR2B9c stability.

TABLE 4 Buffering Species Selection Species pH Pro Con Histidine 6.0Excellent stability, historic use in Chromatographic interference, butlyophilization applications, well within chromatography could possiblybe buffering range improved by new histidine vendor Citrate 6.0 Improvedstability, historic use in lyophilization applications, well withinbuffering range Histidine 6.5 Improved stability, historic use inChromatographic interference, but lyophilization applications, wellwithin chromatography could possibly be buffering range improved by newhistidine vendor Citrate 6.5 Excellent stability, historic use in TargetpH of 6.5 may be on the edge of lyophilization applications, well withinthe ideal buffering range for the citrate buffering range speciesPhosphate 6.5 Improved stability, historic use in Phosphate species hasbeen historically phosphate species in NA-1 formulations avoided forlyophilization formulations

Example 3: Determination of TAT-NR2B9c Stability in Histidine andCitrate Buffers and at Different pH Values with Varying Amounts ofSodium Chloride

The goal of this study was to demonstrate the effects of sodium chloride(NaCl) on pH and TAT-NR2B9c stability in liquid formulations. Bufferformulations with 1 mg/mL TAT-NR2B9c are listed in Table 5, and resultsfor pH are provided in Table 6. The data show fairly consistent resultsfor the duration of the study. However, notable shifts occurred incitrate with the addition of NaCl, where the buffering capacity wasimpacted and the pH dropped by ˜0.2 units. Selected pH's of 6.0 and 6.5are on the outside edge of citrate's ideal buffering range (pH 2.5-5.6),so this may cause difficulties with various additives during thecompounding process and should be considered when evaluating formulationrobustness.

TABLE 5 Buffer formulations for examining the effect of salt on pHVehicle # Buffer Target pH NaCl 1 50 mM Citrate 6.0 NA 2 50 mM Citrate6.0 200 mM 3 50 mM Citrate 6.5 NA 4 50 mM Citrate 6.5 200 mM 5 50 mMHistidine 6.0 NA 6 50 mM Histidine 6.0 200 mM 7 50 mM Histidine 6.5 NA 850 mM Histidine 6.5 200 mM

The results indicate that the addition of 200 mM NaCl to the histidineand citrate buffered TAT-NR2B9c solutions does not significantly affectthe pH of the solutions whether stored for a week frozen or at theaccelerated temperature of 40° C.

Next, we examined the stability of TAT-NR2B9c in these formulations whenstored 1 week at frozen and accelerated temperatures. Table 7 shows theresults of the testing using the RP-HPLC method with an MSA gradient.The data is also presented in FIGS. 2A and 2B. FIG. 2A presents theaccelerated stability of formulations sorted from left to right (low tohigh stability). FIG. 2B shows the relative accelerated stability bybuffering agent.

TABLE 7 Purity (MSA Method), TAT-NR2B9c pH 6.0 pH 6.5 Vehicle −20° C.40° C. −20° C. 40° C. His 98.1 92.8 98.5 96.9 His + NaCl 98.4 95.0 98.497.7 Citr 97.5 96.0 99.0 97.5 Citr + NaCl 98.4 96.7 98.7 98.4

These results indicate that TAT-NR2B9c solution stability is bestmaintained at pH 6.5, and the addition of NaCl may offer a slightimprovement in stability (FIGS. 2A and 2B). Due to improved bufferingcapacity and comparable stability of the histidine buffer, especiallywhen the contaminant migrating with a relative retention time (RRT) of0.28 is excluded (contaminant area included in the table above,resulting in a lower stability value for the TAT-NR2B9c peak area), thehistidine buffering species at pH 6.5 is the best formulation to moveinto lyophilization studies.

Vehicles at pH 6.5 are well tolerated for administration by IV.

Example 4: Selection of Bulking Agents for TAT-NR2B9c to Form a StableLyophilized Cake

To identify bulking agents that would generate a nice cake uponlyophilization and improve stability, we compounded several 20 mg/mLTAT-NR2B9c solutions in 50 mM histidine buffer, bulking agent and NaClas outlined in Table 8. To simulate the time and handling temperaturesthat TAT-NR2B9c formulations may be exposed to during the lyophilizationprocess, these samples were stored at −20° C. (control) and 40° C./75%RH (test), and were analyzed after one week of storage for purity byHPLC (MSA method) and pH. Results are for pH stability are outlined inTable 9, and results for the stability of TAT-NR2B9c in the differentliquid formulations are shown in Table 10 and FIG. 3.

TABLE 9 pH, Bulking Agent Samples Vehicle Target pH pH, −20° C. pH, 40°C. Mannitol 6.5 6.5 6.5 Mannitol + NaCl 6.5 6.5 6.5 Trehalose 6.5 6.56.4 Trehalose + NaCl 6.5 6.5 6.4 Dextran-40 6.5 6.5 6.3 Dextran-40 +NaCl 6.5 6.5 6.4

TABLE 10 Purity by % Area of TAT-NR2B9c Peak, MSA Method % Area of NA-1peak Vehicle # Vehicle −20° C. 40° C. 1 Mannitol 99.2 98.5 2 Mannitol +NaCl 99.4 98.6 3 Trahalose 99.1 98.5 4 Trehalose + NaCl 99.3 98.3 5Dextran-40 99.2 97.6 6 Dextran-40 + NaCl 99.0 97.7

Results of Bulking Agent Liquid Formulations on TAT-NR2B9c Stability

Mannitol, Trehalose and Dextran-40 maintain the pH at 6.5 well (Table 9)and there is approximately a 1% decrease in purity (Table 10) over 1week as a liquid formation when stored at high temperature. In terms ofthe chemical stability of the TAT-NR2B9c lyophilization fill solution,mannitol and trehalose are preferred bulking agents as they conferbetter stability to TAT-NR2B9c than the dextran-40 solutions (FIG. 3).

Example 5: Thermal Analysis of Bulking Agents to Facilitate Design ofLyophilization Cycles

As part of the lyophilization cycle development for TAT-NR2B9clyophilized drug product, proposed fill solutions from the bulking agentsample matrix (Table 8) were evaluated by Differential Scanningcalorimetry (DSC) for thermal characteristics including glass transition(Tg) in the formulation. Results are listed in Table 11 and DSC tracesare included in FIGS. 4A-6B.

TABLE 11 Glass Transitions of TAT-NR2B9c Lyophilization Fill SolutionsVehicle T_(g) 50 mM histidine, pH 6.5, 120 mM Mannitol −37.25° C. 50 mMhistidine, pH 6.5, 120 mM Mannitol, 75 mM NaCl −42.51° C. 50 mMhistidine, pH 6.5, 120 mM Trehalose −28.25° C. 50 mM histidine, pH 6.5,120 mM Trehalose, 75 mM NaCl −35.74° C. 50 mM histidine, pH 6.5, 5%Dextran-40 −17.09° C. 50 mM histidine, pH 6.5, 5% Dextran-40, 75 mM NaCl−22.49° C.

At a TAT-NR2B9c concentration of 20 mg/mL, tested TAT-NR2B9cformulations showed a thermal profile characterized by a broad meltingevent with onset at a low temperature. This extended melt masked thecrystallization event typically seen in mannitol formulations, and mayindicate that a robust freeze drying cycle must be performed where theproduct never exceeds the glass transition temperature. In this case,based on the observed glass transitions of the TAT-NR2B9c drug productfill solution, the use of mannitol as a bulking agent would require aprimary drying temperature lower than −40° C., the typical limit offeasibility for a scalable cycle. In terms of thermal profiles,Trehalose and Dextran-40 are superior for use as a bulking agent.However, given that the stability of TAT-NR2B9c in the liquidformulations containing trehalose was superior to those containingDextran, trehalose would be the preferred bulking agent of those tested.

Due to the relatively low Tg temperatures that would likely require alonger lyophilization cycle to dry, we looked at a wider range ofstandard bulking agents and looked to reduce the fill volume into thecontainer closure system so that there would be a reduced volume ofliquid to lyophilize. In an effort to decrease the fill volume andmaintain 270 mg/vial, a solubility study of TAT-NR2B9c in Histidine, pH6.5 and in Histidine +Trehalose, pH 6.5 was performed. Samples werevisually analyzed at 35, 50, 75 and 100 mg/mL. All solutions were clearat t=0 and t=24 hours. Based on this data, we could use fill volumelower than 3 mL, which using a 90 mg/mL TAT-NR2B9c formulation wouldgive provide 270 mg in a target vial. A wide range of quantities may berequired in a vial, but 270 mg would provide a 2.6 mg/kg dose for a 100kg patient. Assuming the target reconstitution concentration for patientadministration is still 20 mg/mL (but can be from 1 mg/ml to 100 mg/ml),then a 20-mL lyophilization vial containing 270 mg of TAT-NR2B9c can beused with a reconstitution volume of 13.5 mL. Therefore, optimal volumesof liquid for lyophilization of TAT-NR2B9c in the vial would be between2.5 mL and 10 mL.

A wider range of bulking agents were tested prior to advancing intolyophilization development, and the Tg's are shown in Table 12. The 100mg/mL TAT-NR2B9c in histidine, pH 6.5 was also evaluated by DSC and thedata is included in Table 12.

Based on the DSC data in Table 12, there are several formulation optionsfor both active and placebo drug products. In general, formulations 5and 11 are the most promising for the active product with respect to theTg. Any bulking agent may be suitable for use in a placebo product, butFormulation 4 (Mannitol) will have the shortest cycle length ifannealed, and may be the most desirable should the appearance match theactive.

As we determine an optimal active formulation, it is important toconsider solution stability, lyophilization cycle robustness, andchemical stability. Formulation 5 from Table 12 (Trehalose) demonstratedgood solution stability and lyophile chemical stability at acceleratedconditions (data shown subsequently), but requires a longerlyophilization cycle at a fill configuration of 13.5 mL. This longercycle length may not be ideal for commercial manufacture in the future,where a shorter cycle is desirable. Formulation 11 from Table 12(without a bulking agent, at 100 mg/mL TAT-NR2B9c) has a higher glasstransition temperature than Formulation 5, allowing for a warmer,shorter cycle. In addition, a decreased fill volume will significantlyshorten the run time as there will be less ice to sublimate from eachvial.

Example 6: Stability of TAT-NR2B9c with Varying Bulking Agents, Scalesand Lyophilization Conditions Bulking Agent Accelerated Stability

A small batch of TAT-NR2B9c drug product was lyophilized to evaluatesolid state stability after 1 week at 25° C., 40° C., and 60° C.TAT-NR2B9c was compounded at an active concentration of 20 mg/mL inthree different vehicles. Samples were evaluated for appearance,reconstitution, pH, amount and purity by HPLC (MSA method) at t=0 andt=1 week. Water content was evaluated at t=0 only.

All TAT-NR2B9c drug products appeared as white, lyophilized cakes andreconstituted in less than 10 seconds at t=0 and t=1 week.

The drug product vehicles are described in Table 13 and are listed withthe respective glass transition temperature and water content results.The pH, TAT-NR2B9c amount and TAT-NR2B9c purity results are described inTables 14-16.

TABLE 13 Bulking Agent Sample Matrix: Tg, and % Water Content t = 0Vehicle # Vehicle T_(g) % Water Content 1 50 mM His, pH 6.5 + −29.93° C.0.29% 120 mM Trehalose −28.25° C. w/NA-1 2 50 mM His, pH 6.5 + −22.60°C. 0.05% 5% Dextran-40 3 50 mM His, pH 6.5 + −17.09° C. 0.10% 1:1 120 mMw/NA-1 Trehalose:5% Dextran-40

TABLE 14 pH, Bulking Agent Lyo Small Scale #1 Theo- Measured pH reticalt = 1 wk t = 1 wk t = 1 wk Bulking Agent pH t = 0 25° C. 40° C. 60° C.Trehalose 6.5 6.4 6.4 6.4 6.4 Dextran-40 6.5 6.4 6.3 6.3 6.3 1:1 6.5 6.46.3 6.4 6.4 Trehalose:Dextran

TABLE 15 Amount (mg/vial), Bulking Agent Lyo Small Scale #1 t = 1 week t= 1 week t = 1 week Bulking Agent t = 0 25° C. 40° C. 60° C. Trehalose20.6 20.3 20.7 20.7 Dextran-40 19.4 19.8 19.5 19.1 1:1Trehalose:Dextran-40 20.3 20.8 20.2 20.2

TABLE 16 Purity (% Area by HPLC), Bulking Agent Lyo Small Scale #1 t = 1week t = 1 week t = 1 week Bulking Agent t = 0 25° C. 40° C. 60° C.Trehalose 98.8 98.8 98.8 98.4 Dextran-40 98.9 98.9 98.6 96.5 1:1Trehalose:Dextran-40 98.9 98.8 98.6 97.5

All three bulking agents, Trehalose, Dextran-40 andTrehalose:Dextran-40, maintain the pH at 6.5 (Table 14) and there is arange of 0.5-2.5% decrease in purity at 60° C. after 1 week (Table 15).Both drug products containing dextran-40 and stored at 60° C. showedgrowth in related substances at a retention time (RT) ˜6.0. Theserelated substances were not present in the trehalose samples, suggestingthat trehalose has a stabilizing effect in the lyophilized drug productand dextran-40 can cause a specific degradation product.

The inclusion of dextran-40 in the bulking agent allows for a warmerprimary drying temperature, but dextran-40 as a bulking agentdemonstrated the poorest stability. The combination of trehalose anddextran-40 (1:1) results in a glass transition temperature that isapproximately 10° C. warmer than trehalose alone. However, it appearsthat the 60° C. stability is intermediate to the trehalose and dextranalone samples, so that trehalose is a preferred bulking agent.

Lyophilized TAT-NR2B9c Formulation Development: Small Scale Experiment#2

A small batch of TAT-NR2B9c drug product was lyophilized to evaluatesetting the shelf temperature at 5° C. during primary drying. TAT-NR2B9cwas compounded at an active concentration of 27 mg/mL in 50 mMHistidine, pH 6.5 and 120 mM Trehalose. The cycle parameters areoutlined in Table 17. Four 20-mL glass lyophilization vials were filledwith 10 mL. Two vials were probed with temperature probes.

TABLE 17 Small Scale 2, TAT-NR2B9c Formulation Development TemperatureHold/ Rate Time Pressure Function (° C.) Rate (° C./minute) (minutes)(mTorr) Load 5 Hold — 0 Ambient Equilibration 5 Hold — 120 AmbientFreeze −40 Rate 0.5 90 Ambient Freeze −40 Hold — 240 Ambient Primary 5Rate  0.25 180 225 Drying¹ Primary 5 Hold — 2050  50 Drying¹ Secondary25 Rate 0.1 200  50 Drying Secondary 25 Hold — 1440  50 Drying Stopper20 Hold — — Nitrogen/ Ambient Unload 20 Hold — — Ambient ¹Primary dryingtemperature based on large vial size and fill volume, not directlyrelated to glass transition temperature.

Due to the large fill volume, it is necessary to set the shelftemperature considerably warmer than the glass transition temperature inorder to compensate for evaporative cooling. The solution temperatureduring primary drying was at −29° C., which is near the glass transitiontemperature of −28° C. from the DSC thermal analysis.

90 mg/mL TAT-NR2B9c Lyophile Accelerated Stability (Small Scale 3)

Prior to compounding the small scale 3 fill solution, a 90 mg/mLTAT-NR2B9c in buffer (50 mM Histidine, pH 6.5) was evaluated for pH. ThepH of the solution was 6.04. It was determined that with the increasedconcentration of TAT-NR2B9c, the buffering strength also needed toincrease. Solutions were prepared at 150 mM, 100 mM, 75 mM, and waterand evaluated for pH. The pHs are listed in Table 18. Small scale 3 wascompounded in a 100 mM Histidine buffer at pH 6.5, and the pH wasre-adjusted to 6.5 after addition of TAT-NR2B9c.

TABLE 18 pH, 90 mg/mL TAT-NR2B9c in Histidine Buffers, pH 6.5 Buffer pHWater 5.39 50 mM 6.04 75 mM 6.04 100 mM 6.29 150 mM 6.14

A small batch of TAT-NR2B9c drug product was lyophilized to evaluatesolid state stability after 1 week storage at 25° C. and 60° C. Two 90mg/mL TAT-NR2B9c formulations were compounded (buffer and buffer withtrehalose). Samples were evaluated for appearance, reconstitution, watercontent and purity by HPLC (MSA method) at t=0 and t=1 week.

All TAT-NR2B9c drug products appeared as white, lyophilized cakes. Somecakes were cracked. Placebo formulations were visually similar to theactive formulations.

Reconstitution time was approximately 1.5 minutes compared to less than10 seconds in the previous formulations. The increased reconstitutiontime is most likely due to the increased concentrations of TAT-NR2B9cand histidine. Further tests showed good stability of TAT-NR2B9c withhistidine buffer concentrations of 50 and 75 mM, with shortenedresuspension times. Also, due to the 2-mL vial size used for this study,only 1 mL of water was added to the lyophile. The actual reconstitutionvolume is 4.5 mL in this small scale configuration. The reconstitutiontime will most likely improve when a larger volume of diluent is used.

Vials were placed on stability at 25° C. and 60° C. and tested after 1week of storage.

Based on the visual appearance data, the trehalose sample gave a moreelegant cake. The lyophilization cycle was run conservatively over 5days, with a primary drying temperature of −32°. Based on thetemperature probe data, the cycle can be shortened, demonstrating thatwith the higher concentration and lower fill volume the optimized cyclewill be shorter.

Purity results are outlined in Table 19.

TABLE 19 Purity (% Area by HPLC), Small Scale 3 t = 1 week, t = 1 Fill25° C./ week, Formulation Composition Solution t = 0 60% RH 60° C. 1 100mM His, 99.2 99.3 99.3 97.8 pH 6.5 2 100 mM His, 99.2 99.3 99.2 96.7 pH6.5 + 120 mM Trehalose

Based on this accelerated stability data, trehalose demonstrates astabilizing effect on the TAT-NR2B9c formulation which improves thechemical stability of the lyophile. It is surprising that trehalose isable to confer this stabilizing effect while other standard bulkingagents such as dextran and mannitol used for other peptides do not.

The reduced fill volume minimizes the competing evaporative cooling ofthe surrounding vials and minimizes the resistance to the sublimatingwater.

Lyophilization Cycle Development—Small Scale 4 (Placebo and Active)

Small scale 4 of the lyophilization cycle development was initiated totest the cake appearance and lyophilization conditions for a 3 mL fill.Samples tested were 100 mM His, pH 6.5 with 120 mM Trehalose and 90mg/kg TAT-NR2B9c or an identical sample removing the Trehalose. Aconservative, 4 day cycle was ran as described in Table 20. Placebo andactive vials were included with a fill configuration of 3 mL into a20-mL glass lyophilization vial instead of the small vials used for theprevious experiments. An active temperature probe was used to confirmtemperature during the lyophilization cycle. The resulting active vialsis shown in FIG. 7A.

TABLE 20 Lyophilization Parameters for Small Scale 4 Temperature Hold/Rate Time Pressure Function (° C.) Rate (° C./minute) (minutes) (mTorr)Load 5 Hold — 0 Ambient Equilibration 5 Hold — 120 Ambient Freeze −40Rate 0.5 90 Ambient Freeze −40 Hold — 120 Ambient Primary −30 Rate  0.2540 225 Drying Primary −30 Hold — 3400  50 Drying Secondary 25 Rate 0.1550  50 Drying Secondary 25 Hold — 1440  50 Drying Stopper 20 Hold — —Nitrogen/ Ambient Unload 20 Hold — — Ambient

Formulation at 90 mg/ml in a 20 mL vial formed an elegant cake on a 4day cycle, and temperature probe data suggested that the cycle could beshortened to 3 days.

Water content for the placebo and active was 0.01% and 0.00%.

Lyophilization Cycle Development—Small Scale 5 (Placebo and Active)

Small Scale 5 was performed to look at developing a matching placebovial for clinical trials and to look at resuspension times forformulations at a potential commercial scale (270 mg/vial). 10 placeboformulations and 1 active formulation were evaluated for appearance andreconstitution time. The active cakes were elegant, white cakes withminor shrinkage resulting in a crack around the surface of the vialwall. The placebo cakes were white with more cracks in cakes containingincreasing amounts of trehalose.

Vials were reconstituted with 13.5 mL of water. The time to dissolve islisted in Table 21. The active lyophile re-suspended immediately, butwas cloudy for 17.6 sec before becoming a clear, colorless solution. Allplacebos were a clear, colorless solution.

TABLE 21 Reconstitution of Placebo and Active (SS5) FormulationsReconstitution Placebo Trehalose, Histidine, Total, Time (min)Formulation # mM mM mg/vial Vial #1 Vial #2 1 (Control) 120 100 170 <10sec <10 sec 2 200 100 252 <10 sec <10 sec 3 300 100 355 <10 sec <10 sec4 400 100 457 <10 sec <10 sec 5 500 100 560 <10 sec <10 sec 6 120 20 133<10 sec <10 sec 7 200 20 215 <10 sec <10 sec 8 300 20 317 <10 sec <10sec 9 400 20 420 <10 sec <10 sec 10  500 20 523 <10 sec <10 sec ActiveTrehalose, Histidine, NA-1, Formulation # mM mM mg Vial #1 Vial #2 1(Control) 120 100 90 17.6 sec NA

Based on the stability, resuspension times, and lyophilization times, apreferred commercial formulation for TAT-NR2B9c, prelyophilization,would be 20-100 mM Histidine, 120 mM Trehalose pH 6.5. Trehaloseconcentrations can be increased without a loss of stability or cakeelegance but resuspension times.

Examination of Increased Trehalose in Cake Formation and PlaceboMatching by Visual Appearance and Resuspension Time

To better match a placebo, varying concentrations of Trehalose weretested with and without TAT-NR2B9c, and at either 3 or 5 mL fillvolumes.

First, the active formulations and the placebo formulations will besummarized. Then the lead visual matches for the 3-mL fill and the 5-mLfill will be highlighted. Analytical samples (fill solution and onepotency sample) are currently being analyzed. Tables 22 and 23 show asubset of the formulations tested.

TABLE 22 Active Formulations Formulation Fill Volume Composition 1 3-mL270 mg/vial in 120 mM Trehalose + 100 mM Histidine, pH 6.5 2 3-mL 270mg/vial in 500 mM Trehalose + 20 mM Histidine, pH 6.5 3 5-mL 270 mg/vialin 120 mM Trehalose + 50 mM Histidine, pH 6.5

FIG. 7B shows the appearance of the active formulations listed above.

TABLE 23 Placebo Formulations Active Formulations Placebo Formulations(~270 mg/vial) 500 mM Trehalose + 500 mM Trehalose + 20 mM Histidine (n= 7) 20 mM Histidine (n = 2) 400 mM Trehalose + 400 mM Trehalose + 20 mMHistidine (n = 3) 20 mM Histidine (n = 1) 300 mM Trehalose + 300 mMTrehalose + 20 mM Histidine (n = 3) 20 mM Histidine (n = 1)

Table 24 shows the lyophilization cycle conditions for the above samples

TABLE 24 Cycle Parameters Temperature Hold/ Rate Time Pressure Function(° C.) Rate (° C./minute) (minutes) (mTorr) Load 5 Hold — 0 AmbientEquilibration 5 Hold — 120 Ambient Freeze −40 Rate 0.25 180 AmbientFreeze −40 Hold — 120 Ambient Anneal −27 Rate 0.25 52 Ambient Anneal −27Hold — 120 Ambient Freeze −40 Rate 0.25 52 Ambient Freeze −40 Hold — 120Ambient Primary −30 Rate 0.25 40 225 Drying Primary −30 Hold — 4406  50Drying Secondary 25 Rate 0.1  550  50 Drying Secondary 25 Hold — 1440 50 Drying Stopper 20 Hold — — Nitrogen/ Ambient Unload 20 Hold — —Ambient

TABLE 25A Summary of Lead Matches Topography: Color & Ex. Skin, Finish:bumps, Structure: Sample (Sheen or cracks, peak, Dense or ReconstitutionName Matte) curls pourous Shrinkage Friability Time SS6 - 3 mL FillsActive #2 off white thin cracks dense minimal see photo 2 min 30 sec 500mM matte Trehalose 50 mM Histidine Placebo P2 off white cracks denseminimal 1 min 500 mM matte Trehalose 20 mM Histidine SS7 - 5 mL FillsActive off white cracked semi-dense, yes, base of NT 1 min 500 mM mattepocked layered cake Trehalose w/shiny bottom 20 mM specks HistidinePlacebo off white cracked semi-dense, minimal 20 sec 500 mM matte pockedmore porous unannealed: Trehalose w/shiny bottom than active 38 sec 20mM specks Histidine

Example 7: Stability of Lyophilized 270 mg TAT-NR2B9c in 20 mM HistidineBuffer pH 6.5 and 120 mM Trehalose Preparation of the Lyophilized DrugProduct

A small batch of TAT-NR2B9c drug product was formulated at 90 mg/mL in20 mM Histidine pH 6.5 and 120 mM trehalose and lyophilized to evaluatesolid state stability after 4 weeks at −20° C., 40° C., and 60° C. Table25 shows the lyophilization conditions.

TABLE 25B Lyophilization cycle conditions for Example 7 TemperatureHold/ Rate Time Pressure Function (° C.) Rate (° C./minute) (minutes)(mTorr) Load 5 Hold — 0 Ambient Equilibration 5 Hold — 120 AmbientFreeze −40 Rate 0.5 90 Ambient Freeze −40 Hold — 120 Ambient Primary −28Rate  0.25 48 225 Drying Primary −28 Hold — 3412  50 Drying Secondary 25Rate 0.1 530  50 Drying Secondary 25 Hold — 1440  50 Drying Stopper 20Hold — — Nitrogen/ Ambient Unload 20 Hold — — Ambient

Samples were stored in constant temperature ovens with and the purity,potency, and reconstitution time in 13.2 mL (for 13.5 final volume) wereassessed at 0, 1, 2 and 4 weeks. The data for each storage temperatureand time is presented in Tables 26A-C.

TABLE 26A Stability at −20° C. Parameter t = 0 t = 4 weeks AppearanceDense white cake Dense white cake Reconstitution Time ~60 sec ~60 sec pH6.32 TBD Water Content 0.02% NT % Label Claim, TFA Method 99.0% 101.3%Total Purity, MSA Method 99.2% 99.2% (% Area) Individual Impurities RRT% Area RRT % Area 0.59 0.02% 0.59 0.02% ND ND 0.95 0.01% 0.97 0.26% 0.980.21% 1.04 0.26% 1.05 0.32% 1.07 0.09% 1.09 0.04% 1.10 0.13% 1.11 0.12%ND ND 1.14 0.03% 1.15 0.02% 1.16 0.02% Deamidated NA-1, SCX Method<0.05% TBD (% Area)

TABLE 26B Stability at 40° C. Parameter t = 0 t = 1 week t = 2 weeks t =4 weeks Appearance Dense white cake Dense white cake Dense white cakeDense white cake Reconstitution Time ~60 sec ~60 sec ~60 sec ~60 sec pH6.32 6.55 6.21 TBD Water Content 0.02% NT NT NT % Label Claim, TFAMethod 99.0% 97.0% 100.6% 100.6% Total Purity, MSA Method 99.2% 99.1%98.9% 99.0% (% Area) Individual Impurities RRT % Area RRT % Area RRT %Area RRT % Area 0.59 0.02% 0.59 0.02% 0.62 0.02% 0.59 0.02% 0.97 0.26%0.97 0.26% 0.95 0.02% 0.95 0.01% 1.04 0.26% 1.04 0.25% 0.98 0.21% 0.970.17% 1.07 0.09% 1.07 0.13% 1.05 0.33% 1.05 0.27% ND ND ND ND ND ND NDND 1.10 0.13% 1.10 0.15% 1.10 0.17% 1.10 0.19% ND ND 1.13 0.04% 1.130.16% 1.13 0.05% 1.15 0.02% 1.15 0.02% 1.15 0.05% 1.15 ND ND ND ND ND1.17 0.05% 1.16 0.02% 1.26 0.01% 1.26 0.01% 1.29 0.01% 1.28 0.01% 1.290.01% 1.29 0.02% 1.31 0.04% 1.30 0.07% Deamidated NA-1, SCX Method<0.05% NT NT TBD (% Area)

TABLE 26C Stability at 60° C. Parameter t = 0 t = 1 week t = 2 weeks t =4 weeks Appearance Dense white cake Dense white cake Dense white cakeDense white cake Reconstitution Time ~60 sec ~60 sec ~60 sec ~60 sec pH6.32 6.43 6.29 6.29 Water Content 0.02% NT NT NT % Label Claim, TFAMethod 99.0% 97.3% 101.5% 101.5% Total Purity, MSA Method 99.2% 98.8%98.3% 98.0% (% Area) Individual Impurities RRT % Area RRT % Area RRT %Area RRT % Area ND ND 0.53 0.01% 0.53 0.01% 0.51 0.02% 0.59 0.02% 0.590.03% 0.62 0.02% 0.59 0.02% ND ND 0.91 0.01% 0.91 0.02% 0.92 0.01% ND ND0.95 0.02% 0.95 0.02% 0.95 0.03% 0.97 0.26% 0.97 0.26% 0.98 0.25% 0.970.20% 1.04 0.26% 1.04 0.23% 1.05 0.33 1.05 0.28% 1.07 0.09% 1.07 0.24%1.07 ND¹ 1.08 0.59% 1.10 0.13% 1.10 0.23% 1.09 0.37% 1.10 0.44% ND ND1.12 0.05% 1.12 0.30% 1.13 0.09% 1.15 0.02% 1.15 0.02% 1.15 0.10% 1.150.05% ND ND ND ND 1.17 0.07% 1.17 ND 1.26 0.01% 1.26 0.01% 1.26 <0.01%1.27 0.01% 1.29 0.01% 1.29 0.08% 1.30 0.17% 1.30 0.29% Deamidated NA-1,SCX Method <0.05% NT NT TBD (% Area) ¹Loss in resolution around mainpeak

This formulation of TAT-NR2B9c is stable at −20° C. For storagetemperatures of 40° C. and 60° C., potential impurities with relativeretention times (RRT) of 1.07, 1.1 and 1.29 increased slowly using theMSA HPLC assay, with the largest growth appearing at 1.07 RRT. For the40° C. storage temperature, the impurity increases from 0.09% to 0.27%over 1 month, and for the 60° C. storage temperature the impurityincreases from 0.09% to 0.59%. No impurity was observed at -20° C.Impurities interpolated using the Arrnehius equation are less than 0.5%after 16 months at 25° C. or 123 months at 5° C., and less than 2%for >60 months at room temperature and many years at 5° C. Thus, thisand related formulations are suitable for room temperature storage oflyophilized drug product.

This degradation study was allowed to proceed another month to confirmthat the degradation products observed at 60° C. were also apparent at40° C. These three impurities did seem to occur at the lowertemperature, indicating that they are likely to be degradation productsthat are not specific to highly elevated temperatures. The identities ofthese species were determined by LC/MS/MS studies and found to allcomprise acetylation of the full length TAT-NR2B9c compound. Theseacetylation events occurred both at the N-terminus of the peptide and onlysine side chains. Therefore, the stability of TAT-NR2B9c could beincreased by either adding a scavenger or other excipients to reduceacetylation of TAT-NR2B9c in the lyophilized state or by reducing orremoving the acetate so that there is a reduced chance of acetylation.

Overall Conclusions

Based on the stability, resuspension times, and lyophilization times, apreferred commercial formulation for TAT-NR2B9c is 20-100 mM Histidine,120 mM Trehalose pH 6.5. Trehalose concentrations can be increasedwithout a loss of stability or cake elegance but resuspension timesincrease with increased trehalose concentration.

Example 8: Development and Stability of a Chloride Salt of TAT-NR2B9c(TAT-NR2B9c-Cl) Preparation of the Lyophilized Drug Product

Due to the instability of the previously developed saline formulationsof TAT-NR2B9c, and the observation that acetylation of the acetate saltof TAT-NR2B9c can occur in lyophilized formulations, the acetate saltwas exchanged to a chloride salt. This was done by preparative RP-HPLCusing ammonium chloride. The goal of this method is to identify a novelcomposition of matter and a novel formulation that will allow improvedstability for TAT-NR2B9c, preferably with improved stability at bothroom temperature and 37° C. Because TAT-NR2B9c is effective whenadministered to potential victims of stroke and other neurologicaldisorders, and there is a high value to early treatment of thesedisorders, a formulation that is stable outside of hospital conditionswould be valuable in helping the millions of people affected by thesedisorders every year. For example, such a drug could be stored inambulances, or small clinics, doctor's offices or even be available toindividuals, and provided to subjects having neurological disorders suchas stroke or other diseases amenable to neuroprotective agents muchearlier than they might be administered if they had to travel to ahospital for treatment.

General Process Protocol

Buffer A: Water

Buffer B: Acetonitrile

Column: Daisogel ODS C-18 (1Kg), 120 Å, 15 μm, 10 cm (Bed Volume: ˜1L)

Flow Rate: 250 mL/min.

Wavelength: 230 nm

Gradient: Kick out with 20% B

-   -   (1) Column was washed with 2 Bed Volume (BV) 80% CH₃OH    -   (2) Passed 2 BV 0.025% HCl in water or 0.1% TFA in water, which        was superior    -   (3) Loaded sample: 30 g TAT-NR2B9c acetate (PPL-NA11301) was        dissolved in 1.5L USP water (20 g/L) under which conditions,        TAT-NR2B9c binds to the column;    -   (4) Rinsed with 200 mL USP water (also loaded to column)        removing the existing acetate counterion    -   (5) Passed 3 BV 0.1M Ammonium Chloride (NH₄Cl) to supply new        chloride counterion;    -   (6) Passed 2 BV 2%

(7) Passed 20% B (acetonitrile) to elute the product

(8) Collected fractions when the product peak eluted

(9) Fractions were analyzed by Analytical HPLC

(10) Column was back washed with 3 BV 80% CH₃OH

Analytical HPLC System

YMC ODS-A C18 Column, 5 μm, 120 Å; Flow rate: 1.5 mL/min.; Wavelength:210 nm;

Temperature: 50° C.; Gradient: 20% to 35% B in 15 min.; Buffer A=0.1MNaClO₄ pH 3.1,

Buffer B=100% ACN

Synthetic Results and Discussions

Two main pools collected from the runs were mixed and lyophilized (˜5L).23.4 g of the final product was collected, with the purity 99.01%. Theresidual acetate was less than 1%. Standard refinements of the protocolshould be able to increase the yield from 78% to >95%. This procedurecan similarly be used to exchange a TFA salt of TAT-NR2B9c to a chloridesalt, as TFA salt will bind the column in step 3 similarly.

Formulation of a Lyophilized Form of TAT-NR2B9c-Cl and Stability Testing

TAT-NR2B9c-Cl was compounded in 20 mM histidine and 120 mM trehalose, pH6.5, for direct comparison to a previous preferred lyophilizedformulation under the same lyophilization conditions at 270 mg/vial(active weight). The stability of this formulation was also compared tothe previously disclosed formulation of TAT-NR2B9c-Ac resuspended insaline and lyophilized. For the latter, a small batch ofTAT-NR2B9c-acetate drug product was formulated at 90 mg/mL in saline (noother excipients) and lyophilized at 270 mg/vial. Stability of theformulations in the lyophilized state was evaluated after 4 weeks at−20° C. (control), 40° C. and 60° C.

TABLE 27 Stability of TAT-NR2B9c-Cl (20 mM histidine, 120 mM trehalose)versus TAT-NR2B9c-Ac (saline) NA-1 Chloride salt stability data NA-1-AcSALINE stability data RP-HPLC TFA 0.899 0.11 RP-HPLC TFA 0.899 0.95 0.950.11 0.97 0.07 0.97 0.09 0.98 0.98 0.08 0.99 0.17 0.29 0.43 0.99 0.180.23 0.19 1 98 97.78 96.64 1 97.93 97.42 94.84 1.02 1.27 1.22 1.36 1.021.22 1.39 2.07 1.03 0.11 0.11 0.16 1.03 0.1 0.1 0.12 1.05 0.08 0.07 0.091.05 0.05 0.16 0.61 1.06 1.06 0.1 1.07 0.22 0.24 0.46 1.07 0.2 0.19 0.281.08 1.08 0.1 1.09 0.06 1.09 0.07 1.1 0.11 0.09 1.1 0.12 1.11 0.16 0.091.11 0.05 0.06 0.14 1.12 0.11 1.12 0.17 0.17 0.31 1.13 0.06 1.13 0.050.28 0.72 1.14 0.09 1.14 0.06 1.18 1.18 1.19 1.19 1.26 1.26 1.34 1.341.36 1.36 1.4 1.4 1.48 1.48 RP-HPLC-MSA 0.94 0.07 RP-HPLC-MSA 0.94 0.050.98 0.11 0.11 0.15 0.98 0.06 0.07 0.08 1 99.3 99.14 98.68 1 99.13 98.8996.38 1.02 1.02 1.03 0.27 0.31 0.3 1.03 0.41 0.32 0.42 1.04 0.07 0.060.09 1.04 0.09 0.09 1.05 0.25 0.26 0.55 1.06 0.23 0.21 0.22 1.07 0.120.09 1.07 0.08 0.21 1.17 1.08 1.08 1.1 0.07 1.1 0.1 0.59 1.12 1.12 0.071.14 1.14 0.1 1.16 1.16 0.06 1.2 1.2 0.06 0.31 1.23 1.23 1.24 1.24 1.271.27 1.3 1.3 0.06 0.54

TABLE 28 Stability of TAT-NR2B9c-Cl (20 mM histidine, 120 mM trehalose)versus TAT-NR2B9c-Ac (saline) at 11 months NA-1 Chloride salt NA-1-Ac insaline Peak T = 11 months T = 11 months (RRT) −20 C. 40 C. 60 C. −20 C.40 C. 60 C. RP-HPLC- 0.899 0.1 TFA 0.93 0.18 0.95 0.16 0.27 0.97 0.10.17 0.99 0.29 0.38 0.22 1 98.57 98.14 95.49 98.57 96.48 81.27 1.02 1.181.09 1.42 1.14 1.8 6.51 1.03 0.07 0.09 0.34 0.09 1.05 0.05 0.17 0.51 31.06 0.15 0.06 0.27 1.07 0.18 0.36 1.05 0.2 0.27 0.62 1.08 0.08 0.3 1.090.16 1.1 0.06 0.28 1.11 0.29 1.12 0.12 0.33 1.13 0.41 3.49 1.15 0.421.16 0.16 1.17 0.26 1.18 0.1 1.19 0.23 1.2 0.26 1.22 0.19 1.23 0.3 1.240.25 1.26 0.34 1.261 0.34 1.34 0.14 RP-HPLC- 0.52 0.13 MSA 0.87 0.130.91 0.05 0.2 0.23 0.94 0.09 0.34 0.29 0.98 0.1 0.12 0.21 0.1 0.12 0.1 199.3 98.71 95.34 99.32 97.08 82.12 1.02 0.29 0.24 0.61 0.24 0.28 0.821.04 0.07 0.12 0.08 0.11 1.06 0.25 0.48 1.48 1.07 0.06 0.25 0.25 1.036.36 1.09 0.08 0.26 0.45 3.02 1.1 0.27 1.12 0.05 0.23 1.14 0.17 0.311.15 0.53 1.16 0.11 0.26 1.19 0.33 1.2 0.1 1.23 0.05 0.24 0.1 0.59 1.240.06 0.59 1.25 0.18 0.35 1.26 0.05 0.44 1.27 0.41 3.18 1.33 0.26 1.350.15

Table 27 shows the relative area of each peak observed in each HPLCassay at each temperature after 1 month of storage and at each relativeretention time (peak identity) for direct comparison. For bothformulations and methods, the starting purities are very similar (98%for TAT-NR2B9c-Cl vs 97.93% for TAT-NR2B9c-Ac by the TFA method, and99.3% vs 99.13% for the methanesulfonic acid (MSA) method. Looking atthe 60° C. data, the TAT-NR2B9c-Cl histidine trehalose composition issubstantially more stable than the TAT-NR2B9c saline formulation by bothmethods. For the TFA method after 1 month of storage at 60° C., theTAT-NR2B9c-Cl salt retains 98.6% of the main peak whereas theTAT-NR2B9c-Ac saline formulation only retains 96.8% of the startingmaterial. For the MSA method, the difference is even more striking,where the TAT-NR2B9c-Cl salt retains 99.4% of its starting materialwhereas the TAT-NR2B9c-Ac saline formation only retains 97.2% of itsstarting material.

Table 28 shows the relative area of each peak observed in each HPLCassay at in the same samples after 11 months at the different storagetemperatures. These data support the improved stability of the NA-1 Clsalt formulation versus the NA-1-Ac formulation in chloride. Comparingthe area of the main NA-1 peaks in the TFA assay at 40° C., which is theaccelerated condition by ICH guidance to support room temperaturestorage of a lyophilized drug, the NA-1-Cl retains 98.14% purity from astarting purity of 98.58, while the NA-1-Ac in saline has dropped from98.57 pure to 96.48, which is below the purity specification of 97% andwould not be considered stable after about a year. The same trend isobserved for the MSA assay from 99.3% pure to 98.71 pure for NA-1-Cl and97.08 for NA-1-Ac in saline. Examining the 60° C. purity results after11 months (Table 28), the differences are significantly more profound,with NA-1-Cl being significantly more stable than NA-1-Ac

From a clinical standpoint, example purity limits for a drug for humansmay be 97% purity with no uncharacterized impurity over 0.5% at therated storage condition. Using the Arrenhius equation, we can use the40° C. and 60° C. stability data to predict the drop in purity of themain peak or the growth of the observed impurities at different storagetemperatures (e.g., 25° C. or 37° C. versus the observed 40° C. or 60°C. data). Assuming that the drug would be stored at ambient temperature,it would be useful to demonstrate stability at 37° C. because therewould be many environments without refrigeration where the ambienttemperature in the summer could be in this range. Assuming a purityrequirement of 97.0% at 37° C., and using the representative stabilitydata from the TFA assay for main peak purity because it is the mostsensitive (i.e., shows the biggest decline in purity at 40° C. and 60°C.), the saline formulation would be predicted to have a shelf life of29.8 months (or a little over 2 years), whereas the TAT-NR2B9c-Clformulation would have a shelf life of ˜500 months, or approximately 41years. This is a significant improvement in stability.

From the standpoint of the growth of the largest impurity (aside fromthe impurity at relative retention time (RTT) 1.02 by the TFA methodwhich is an identified impurity), the largest impurity growth after 1month for the TAT-NR2B9c-Cl formulation is 0.17 to 0.43 (RRT 0.99 by theTFA method), whereas the largest impurity growth for the TAT-NR2B9c-Acsaline formulation is 0.05-0.72 (1.13 RRT by the TFA method). Assumingthe impurity limit above as 0.5% at 37° C., and using the Arrenhiusequation along with the 40C and 60C data from each of these impurities,the saline formulation is predicted to have a shelf life of ˜4.8 monthsat 37° C. whereas the shelf life of the TAT-NR2B9c-Cl composition ispredicted to be >10 years. Thus, from either the standpoint of the totallevel of TAT-NR2B9c or the growth of the largest impurity, theTAT-NR2B9c-Cl composition is a significant improvement over the salineformulation.

Example 9: TAT-NR2B9c-Cl is More Stable Than TAT-NR2B9c-Ac in Histidine,Trehalose Formulations

TAT-NR2B9c-Cl and TAT-NR2B9c-Ac were both formulated at 90 mg/mL in 20mM Histidine, 120 mM trehalose pH 6.5. Three milliliter aliquots (270mg) of each were lyophilized under the same program as presented supra,and on completion were placed into constant temperature and humidityincubators at −20° C. (control), 40° C. and 60° C. After 4 weeks, thestability of each formulation was tested using two orthogonal HPLCmethods—one with TFA as the carrier and one with MSA as the carrier.

Table 29 shows the relative area of each peak observed in each of thetwo HPLC assays at each temperature after 1 month of storage and at eachrelative retention time (RRT; peak identity) for direct comparison. Theleft panel represents the TAT-NR2B9c-Ac formulation and the right panelshows the results of the TAT-NR2B9c-Cl salt formulation. The peak at anRRT of 1 corresponds to the intact TAT-NR2B9c peptide in each assay andcolumn. For both formulations and methods, the starting purities arevery similar (98% for TAT-NR2B9c-Cl vs 98.3% for TAT-NR2B9c-Ac by theTFA method, and 99.3% vs 99.2% respectively for the MSA method). Lookingat the 60° C. data, the TAT-NR2B9c-Cl composition is substantially morestable than the TAT-NR2B9c-Ac formulation by both methods. For the TFAmethod after 1 month of storage at 60° C., the TAT-NR2B9c-Cl saltretains 98.6% of the main peak (96.64% divided by 98% starting) whereasthe TAT-NR2B9c-Ac saline formulation only retains 96% of the startingmaterial (94.44% divided by 98.3% starting purity). Form the MSA method,the difference is even more striking, where the TAT-NR2B9c-Cl saltretains 99.4% of its starting material (98.68%/99.3%) whereas theTAT-NR2B9c-Ac saline formation only retains 95.6% of its startingmaterial (94.81%/99.2%). When one looks at the largest impurities foreither composition, it is clear that for both the 40° C. and 60° C.accelerated stability conditions that the TAT-NR2B9c-acetate salt haslarger impurities and is thus less stable. The chloride salt ofTAT-NR2B9c is surprisingly more stable than the acetate salt underidentical conditions.

Table 30 shows the relative area of each peak observed in each HPLCassay at in the same samples after 11 months at the different storagetemperatures. These data support the improved stability of the NA-1 Clsalt formulation versus the NA-1-Ac salt when formulated in the exactsame buffer (20 mM Histidine/120 mM Trehalose pH 6.5). Comparing thearea of the main NA-1 peaks in the TFA assay at 40° C., which is theaccelerated condition by ICH guidance to support room temperaturestorage of a lyophilized drug, the NA-1-Cl retains 98.14% purity from astarting purity of 98.58, while the NA-1-Ac in saline has dropped from98.57 pure to 95.91, which is below the purity specification of 97% andwould not be considered stable. The same trend is observed for the MSAassay from 99.3% pure to 98.71 pure for NA-1-Cl and 96.85% for NA-1-Acin saline. Examining the 60° C. purity results after 11 months (Table30), the differences are significantly more profound, with NA-I-Cl beingsignificantly more stable than NA-1-Ac (˜95% pure for both assays forthe chloride salt vs ˜83% pure for the acetate salt). Thus, the NA-1chloride salt is predicted to be surprisingly and significantly morestable than the acetate salt in the same buffer.

TABLE 29 Stability of TAT-NR2B9c-Ac versus TAT-NR2B9c-Cl lyophilized in20 mM Histidine 120 mM Trehalose pH 6.5 at 4 weeks T = 4 weeks T = 4weeks RRT −20 C. 40 C. 60 C. RRT −20 C. 40 C. 60 C. RP-HPLC TFA 0.86RP-HPLC TFA 0.86 0.88 0.88 0.9 0.9 0.11 0.93 0.93 0.95 0.95 0.97 0.060.97 0.07 0.98 0.98 0.99 0.58 0.56 0.99 0.17 0.29 0.43 1 97.19 94.44 198 97.78 96.64 1.02 1.52 2.18 1.02 1.27 1.22 1.36 1.03 0.12 0.13 1.030.11 0.11 0.16 1.04 0.15 0.71 1.04 1.05 1.05 0.08 0.07 0.09 1.06 0.070.17 1.06 1.07 0.23 0.36 1.07 0.22 0.24 0.46 1.08 0.16 1.08 1.09 0.151.09 0.06 1.1 0.18 1.1 0.11 0.09 1.11 0.21 1.11 0.16 0.09 1.12 1.12 0.111.13 0.09 0.75 1.13 0.06 1.14 1.14 0.09 1.26 1.26 1.34 1.34 1.36 1.361.4 1.4 1.48 1.48 RP-HPLC-MSA 0.77 RP-HPLC-MSA 0.77 0.94 0.09 0.94 0.070.98 0.12 0.19 0.98 0.11 0.11 0.15 0.99 0.7 0.6 0.99 1 98.11 94.81 199.3 99.14 98.68 1.02 0.34 0.38 1.02 1.03 0.1 0.17 1.03 0.27 0.31 0.31.04 1.04 0.07 0.06 0.09 1.06 0.4 1.46 1.06 0.25 0.26 0.55 1.07 1.070.12 0.09 1.08 0.14 0.64 1.08 1.1 0.17 1.1 0.07 1.12 0.14 1.12 1.13 0.061.13 1.14 1.14 1.15 0.19 1.15 1.16 1.16 1.17 0.09 1.17 1.18 0.07 1.181.2 1.2 1.23 0.17 1.23 1.24 0.18 1.24 1.27 0.08 0.59 1.27 1.3 1.3

TABLE 30 Stability of TAT-NR2B9c-Ac versus TAT-NR2B9c-Cl lyophilized in20 mM Histidine 120 mM Trehalose pH 6.5 after 11 months NA-1 250 mM His,120 mM Trehalose pH 6.5 NA-1 Chloride salt stability data Peak T = 11months T = 11 months (RRT) −20 C. 40 C. 60 C. −20 C. 40 C. 60 C. RP-HPLCTFA 0.86 0.12 0.52 0.88 0.17 0.899 0.15 0.1 0.93 0.18 0.95 0.14 0.160.97 0.19 0.1 0.99 0.37 0.63 0.29 0.38 1 98.47 95.91 83.08 98.57 98.1495.49 1.02 1.21 1.93 5.77 1.18 1.09 1.42 1.03 0.08 0.07 0.09 0.34 1.050.05 0.57 3.45 0.05 0.17 1.06 0.07 0.19 0.15 1.07 0.19 0.31 0.75 0.180.36 1.05 1.08 0.11 0.39 1.09 0.07 0.27 1.1 0.07 0.31 1.12 0.06 0.290.12 1.13 0.41 2.5 1.15 0.19 1.17 0.2 1.2 0.11 1.22 0.12 1.23 0.12 1.250.12 NA-1 250 mM His, 120 mM Trehalose pH 6.5 NA-1 Chloride saltRP-HPLC-MSA 0.77 0.08 0.48 0.8 0.14 0.87 0.13 0.91 0.15 0.05 0.2 0.940.05 0.14 0.09 0.34 0.945 0.22 0.98 0.11 0.23 0.85 0.1 0.12 0.21 1 99.2396.85 82.73 99.3 98.71 95.34 1.02 0.32 0.35 0.77 0.29 0.24 0.61 1.040.08 0.11 0.77 0.07 0.12 1.06 0.26 1.07 5.05 0.25 0.48 1.48 1.07 0.060.25 1.09 0.56 3.42 0.08 0.26 1.1 0.08 0.41 1.12 0.07 0.32 1.14 0.280.17 1.15 0.09 0.32 1.16 0.11 1.19 0.27 1.23 0.07 0.37 0.05 0.24 1.240.22 1.25 0.18 1.26 0.19 1.27 0.38 2.47 1.35 0.12

1. A chloride salt of a peptide which is TAT-NR2B9c (SEQ ID NO:6) ordiffers from TAT-NR2B9c by up to 5 amino acid substitutions, insertionsor deletions.
 2. The chloride salt of claim 1, which is TAT-NR2B9c. 3.The chloride salt of claim 1, prepared by exchanging trifluoroacetatefor chloride in a trifluoroacetate salt of TAT-NR2B9c.
 4. The chloridesalt of claim 1, prepared by exchanging trifluoroacetate for acetate andthen acetate for chloride starting from a trifluoroacetate salt ofTAT-NR2B 9c .
 5. The chloride salt of claim 1, wherein greater than 99%of anions in the salt are chloride.
 6. A prelyophilized formulationcomprising the chloride salt as defined in claim 1, a buffer and asugar.
 7. The prelyophilized formulation of claim 6, wherein thechloride salt is a chloride salt of TAT-NR2B9c.
 8. The prelyophilizedformulation of claim 6, wherein the buffer is histidine and the sugar istrehalose and the pH is 6-7.
 9. The prelyophilized formulation of claim6 wherein acetate and trifluoroacetate each comprise less than 1% ofanions by weight in the formulation.
 10. The prelyophilized formulationof claim 6, wherein acetate and trifluoroacetate each comprise less than0.1% by weight of anions in the formulation.
 11. The prelyophilizedformulation of claim 6, wherein the chloride salt of the peptide is at aconcentration of 70-120 mg/ml, the histidine is at a concentration of15-100 mM, and the trehalose is at a concentration of 80-160 mM.
 12. Theprelyophilized formulation of claim 11, wherein the chloride salt of thepeptide is at a concentration of 70-120 mg/ml, the histidine is at aconcentration of 20-100 mM, and the trehalose is at a concentration of100-140 mM.
 13. The prelyophilized formulation of claim 6, wherein theTat-NR2B9c is at a concentration of 70-120 mg/ml, the concentration ofhistidine 20-50 mM, and the concentration of trehalose is 100-140 mM.14. The prelyophilized formulation of claim 6, wherein the concentrationof histidine is 20 mM and the concentration of trehalose is 100-200 mM,preferably 120 mM and the concentration of TAT-NR2B9c is 90 mg/ml.
 15. Alyophilized formulation prepared by lyophilizing the prelyophilizedformulation of claim
 6. 16. The lyophilized formulation of claim 15,wherein acetate and trifluoroacetate each comprise less than 1% byweight of anions in the formulation.
 17. The lyophilized formulation ofclaim 15, wherein acetate and trifluoroacetate each comprise less than0.1% by weight of anions in the formulation.
 18. A reconstitutedformulation prepared by combining the lyophilized formulation of claim15 with an aqueous solution.
 19. The reconstituted formulation of claim18, wherein the aqueous solution is water or normal saline.
 20. Thereconstituted formulation of claim 18, wherein the volume of thereconstituted formulation is 3-6 times the volume of the prelyophilizedformulation.
 21. A reconstituted formulation comprising a chloride saltof TAT-NR2B9c at concentration of 15-25 mg/ml, a buffer and a sugar. 22.The reconstituted formulation of claim 21, wherein the buffer ishistidine at a concentration of 4-20 mM and the sugar is trehalose at aconcentration of 20-30 mM and the pH is 6-7.
 23. The reconstitutedformulation of claim 22 wherein acetate and trifluoroacetate eachcomprise less than 1% by weight of anions in the formulation.
 24. Thereconstituted formulation of claim 22, wherein acetate andtrifluoroacetate each comprise less than 0.1% by weight of anions in theformulation.
 25. A method of preparing a formulation, comprising storinga lyophilized formulation sample according to claim 6 for at least aweek at a temperature of at least 20° C.; and reconstituting thelyophilized formulation.
 26. The method of claim 25, wherein thelyophilized formulation is reconstituted in water.
 27. The method ofclaim 25, wherein the lyophilized formulation is reconstituted insaline.
 28. The method of claim 25, further comprising administering thereconstituted formulation, optionally after further dilution in normalsaline, to a patient.
 29. The method of claim 28, wherein thelyophilized formulation is stored for at least a year.
 30. The method ofclaim 29, wherein the storage is at ambient temperature.
 31. The methodof claim 30, wherein the storage includes periods in which thetemperature exceeds 37° C.
 32. The method of claim 28, wherein thepatient has stroke or traumatic injury to the CNS.
 33. The method ofclaim 28, wherein the lyophilized formulation is stored in an ambulance.34. The method of claim 28, wherein the patient has a subarachnoidhemorrhage.
 35. The method of claim 25, wherein the patient isundergoing endovascular repair for an aneurysm.